Software Plan Issues
Contents
1. You can cause problems for yourself if you try this without Knowing what you are doing Contact your local friendly technician for assistance 1 Set up Kohler illumination see section 1 4 1 2 Engage the correct phase annulus for the objective Remove an eyepiece and look into the microscope The phase telescope is in the ocular tube just below the eyepieces rotate the barrel from the 0 position to the CT position You should see a bright ring the result of the phase slit in the condenser assembly Superimposed on a dark ring the result of the phase ring in the objective 3 Bring the bright ring and dark ring into focus using the focus wheel on the ocular barrel NOT the condenser or stage focus It is possible to use these objectives in brightfield simply select the BF position in the condenser assembly The electronic version of this document is the latest version It is the responsibility of the individual to ensure that any paper material is the current version Printed material is uncontrolled documentation Last Revised 13 October 2014 Page 10 of 31 Title SG52 Microscopes Department STEP Document No OP EQP LS 46 Revision A 1 4 If necessary use the centering screws on the condenser assembly to centre the phase contrast ring so that the bright ring overlaps the dark ring symmetrically 5 Rotate the barrel back to the normal position That would be the 0 position 6 Widen the field iris diaphragm op2. This is how Olympus does theirs Figure 1 9 A 10x objective Numerica Aperture agnification Special Remark 10x 0 30 Ph1 00 FN26 5 Correction Field Factor Coverslip Number Correction Figure 1 10 What all those numbers mean Some objectives are adjustable to optimise for different refractive indices of the immersion fluid or variations in coverslip thickness e Magnification Magnification Page 17 of 31 Title SG52 Microscopes Department STEP Document No OP EQP LS 46 Revision A 1 e Numerical Aperture A measure of the light gathering power of the objective e Special Remarks In this case this objective may be used in phase contrast mode and it stipulates we use the Phi phase annulus in the condenser assembly e Correction Factor This objective is designed to assume the microscope is working with parallel beams e Coverslip Correction If a number is specified that limits us to using a coverslip of that particular thickness A range may also be specified e Field Number This gives you the size of the field of view Divide the FN by magnification to obtain the diameter of the fov in mm 1 7 1 Using an immersion objective First of all immersion oils can be toxic it s not nice to have the on your fingers and smeared out over surfaces The next first of all is to use a new clean cover slip and gloves Immersion fluids can be great solvents so fingerprints and other dirt will disso
3. command in the sub dialog that is opened with the arrow adjacent to the Label box e Active ROIS This list displays the current ROIs Clicking on the check box deactivates individual ROIs Such ROls are not displayed e Tools These buttons activate different drawing tools and close the dialog box ROIs are drawn with the left mouse button using the first six tools The electronic version of this document is the latest version It is the responsibility of the individual to ensure that any paper material is the current version Printed material is uncontrolled documentation Last Revised 13 October 2014 Page 26 of 31 Title SG52 Microscopes Department STEP Document No OP EQP LS 46 Revision A 1 With the right mouse button the two ends of polygon defining lines are connected to generate closed polygons the drawing command is terminated and the dialog box reopens fed Polygon Mark The corner points of a polygon straight lines then connect the corners amp Interpolating polygon Mark the corner points of a polygon and the software automatically generates a perimeter curve around it 2 Freehand polygon Delineate the ROI by marking its boundaries by continuously dragging the mouse cursor Ol Ellipse Mark The center of the ellipse and adjust size and form by dragging the mouse Additionally press the Shift key to draw a circle After releasing the left mouse button the position can be changed By clicking ane
4. distances Its very easy to damage the objectives Some of them are sprung to absorb collisions but relying on this should really be a last resort Knowing the working distance will allow you to get into focus quickly and easily Inscribing a small x onto the cover slip or specimen with a diamond scribe or fine point marker may help you identify the focal plane Don t forget that the high magnification lenses require your sample to be upside down In this case mounting a coverslip is a must Know how to clean an objective We can teach you how to clean optics but better if we do it for you but the best practice is to avoid them getting dirty in the first place Do not touch any optics Do not blow on optics to clean them 1 You will know if you done this correctly when you see all the pretty colours The electronic version of this document is the latest version It is the responsibility of the individual to ensure that any paper material is the current version Printed material is uncontrolled documentation Last Revised 13 October 2014 Page 13 of 31 Title SG52 Microscopes Department STEP Document No OP EQP LS 46 Revision A 1 Clean dry air from the wall is best Cans of compressed air can mark surfaces if used incorrectly The filters cannot be cleaned do not attempt it In some cases the filter will operate better with that fingerprint than the cleaning process that removes some of the layers 1 6 Some
5. for illumination of a particular wavelength as follows 1224 N Aopjt N Acon So in air where the N A cannot be greater than 1 the simplest system using illumination of 400nm results in a resolution of 244nm Using oil the N A can be driven up to 1 5 resulting in a resolution of 170nm T Focal Plane This is the point where things are in focus erm that s kind of it really 5 Actually some of them can be cleaned but it is tricky contact a professional 2 We shan t go into it here perhaps in a later chapter if you are interested I Despite having all of these things Royal Rife designed and built incredible microscopes which didn t work The electronic version of this document is the latest version It is the responsibility of the individual to ensure that any paper material is the current version Printed material is uncontrolled documentation Last Revised 13 October 2014 Page 14 of 31 Title SG52 Microscopes Department STEP Document No OP EQP LS 46 Revision A 1 Working distance The working distance is the distance from the end of the objective to the focal plane It s very important to know your working distance as it allows you to plan for how you mount your sample It also helps prevent you crashing the objective into the stage and causing damage Generally the higher the magnification the smaller the working distance A 4x objective will typically have a working distance of sever
6. is required to correct for chromatic and spherical aberration These objective lenses have to be tuned by hand it all adds up to some very expensive optics Figure 1 8 Cutaways of Objective Lenses Small features can still be resolved but the quality of the image will not be great The electronic version of this document is the latest version It is the responsibility of the individual to ensure that any paper material is the current version Printed material is uncontrolled documentation Last Revised 13 October 2014 Page 15 of 31 Title SG52 Microscopes Department STEP Document No OP EQP LS 46 Revision A 1 Here are some scandalously concise notes on aberrations Chromatic Aberration Refractive index in a material is a function of wavelength As light travels through a material different colours will have different path lengths This will be seen as separation of colours in the image Spherical Aberration This will be observed as a halo surrounding an object For a point source it will appear as a symmetrical circular halo surrounding the point It is caused by path length differences caused by refraction of light from the edges of the lens This is usually prevented by stopping down an aperture to prevent off axis rays Astigmatism This manifests itself as different axes having different focal points i e horizontal features not being in focus while vertical features are This can be caused by design or manufa
7. is uncontrolled documentation Last Revised 13 October 2014 Page 30 of 31 Title SG52 MICROSCOPES Department STEP Document No OP EQPILS 4 Revision a1 REVISION CONTROL Revision Last Quality Control Change Number Modification Revision Reviewer ME 22 09 2014 Aur lie Cou don Barry O Connell OP EQP LS 46 Original document release A 1 13 10 2014 Aurelie Couedon Barry O Connell OP EQP LS 46 Section Power up down procedure Coo PS PS PS PR AS YOS Page 31 of 31
8. the measurement commands is called you have to select the ROIs to be measured by mouse click If no ROIs are drawn the start of any of the commands will automatically open the Define ROIs dialog described in the previous chapter 1 Area Measure gt RO gt Area Use this command to measure the area of a Region of Interest The line that defines the border of a ROI is counted as part of it The area unit depends on the image calibration see Chapter 7 1 of user manual Image Calibration 2 Perimeter Measure gt RO gt Perimeter The area unit depends on the image calibration see Chapter 7 1 of the user manual Image Calibration 3 Average Grey Value Measure gt RO gt Average Value In all cases the value will be displayed in a spreadsheet that is generated automatically which can be exported to Excel for manipulation 2 4 3 Background Subtraction Background intensity is unavoidable in wide field fluorescence spectroscopy Working in a dark room can eliminate environmental light but there will always be some unwanted light that reaches the camera Causes are imperfect filter sets and the scattering of light at interfaces the cover slip and the specimen Background intensity reduces the contrast of the image and consequently background subtraction should be a routine operation Subtracting the Image Background Open the Background dialog window with the Background button or via Process gt Background Subtraction No
9. your image to your designated folder in your preferred format jpeg tiff png 9 To change the properties style and size of the bar and the dimension units of the scale bar go to image scale bar scale bar properties 10 For length and area measurements select the Measurements tab or go to Measurements measurements bar to open the measurements button bar Sample features include vertical line E and horizontal line H When the measurements are drawn they can be saved onto your image There is a range of measurement features Note see Cell R manual for more detail 2 2 Time Lapse Experiments 1 Open create your database To create a database select Database Menu and click on the command New Database Type a name in the Database Name field click Next and select Finish 2 Open experiment manager Click on the icon in the toolbar or via the menu using Acquisition and Experiment Manager 3 Most tools can be selected from the standard command toolbar 4 Tocreate an experiment simply select the image acquisition icon 2 and left click into the centre of the experiment plan On the right hand side you will see 3 tabs with Image Frame and Display Under Image you can select image type which is your selected fluorescence filter DIC brightfield light intensity objective and exposure time Under Frame you can select binning 1 2 4 8 As you increase binning resolution is reduced and sensitivity is increased With display you can selec
10. Title SG52 MICROSCOPES Department STEP Document No OP EQP LS 46 Revision A1 Standard Operating Procedure SG52 MICROSCOPES Page 1 of 31 Title SG52 Microscopes Department STEP Document No OP EQP LS 46 Revision A 1 Table of Contents THE TEAS Sacce a O a Ea ERa Eaa aaa 3 1 1 CRONO RULE eina cn rT E a N a a 3 1 2 M120 BURNER OPERATION ia stades a a a A ia s 3 1 3 ANATOMY OF A MICROSCOP ina dance natation D ia aa 4 1 3 1 Transmission Light Source Halogen sisi 5 1 3 2 Reflection Light Source Arc Burner inserer 5 1 3 3 TT SUC STON HORS 7 ULES CUS k nE E a tn et an 5 1 3 4 LZ CY ET AOE Ee Te Le Ce eT ke ee ees See 5 1 3 5 Condenser Assembly and Aperture Stop ss NQMMmmsccscsafbesssccccsenssssesscccseecssssssscceees 5 1 3 6 E1 1 AOE EIE cece D A as ance A A A E AEE ecole 5 L37 OE LS T aE E A a a TR 5 1 3 8 E a EEEE a NEE OIE E EEA cd 5 1 3 9 LO ee ee eT a E rm Tree ree re reo 5 Poa COIS ies soars csc a ss es seats eto sts seas ces eee oso se ee 5 BASIC SR ee eee a re 6 1 4 1 Power UP LOW Procedu Cersan aaa e a a ee need ER S 6 1 4 2 K hler IUuminanon Bretelles M acs sae Ea aS AAE TR 6 1 4 3 EDP COS OE ENES SNE ETT e AN AOPE P A N PEE EE E E ea eee 7 1 4 4 DORA UD E T E AE dede O SR ae en a ll 8 1 4 5 PT DSS CONS en NEN io 9 1 4 6 Diferentalimeri rence CONITOSE gag piece ts Mas nn acalebaukessnsudtasae ind reine en ste nt nue 11 L3 MICRO COTE CARE rennen eer a S a nt tetera et ee te
11. al millimetres but a 100x objective will be in the tens of microns For example if the working distance is 0 3mm then you cannot focus through a 1mm glass slide It would be more sensible to use a coverslip or turn your sample upside down being careful not to contaminate the objective with your sample Expensive objectives with extra long working distances are available Depth of Field The depth of field is the thickness of the focal plane A large depth of field means that there is a large distance between the nearest and farthest objects that are in focus The smaller the aperture the greater the depth of field but a small aperture lowers _max thus lowering the N A Diffraction Limit This is a fundamental limit to what you can resolve As a general rule of thumb if its smaller than the wavelength of light you won t be able to see it Typically features smaller than 700nm will not be resolved properly Of course there are some fluorescence techniques that allow you to see objects smaller than this because they emit light rather than scattering it 1 6 2 Aberrations Because it is impossible to obtain perfect lenses imaging errors may show up They are rare enough with quality modern optics which have corrections designed in but if you are working at the limits of the microscope they can still show up A comparison between objective lenses is shown in Fig 1 8 One lens has 3 elements whilst the other has 10 which is what
12. ariser are shown in Fig 1 6 The electronic version of this document is the latest version It is the responsibility of the individual to ensure that any paper material is the current version Printed material is uncontrolled documentation Last Revised 13 October 2014 Page 11 of 31 Title SG52 Microscopes Department STEP Document No OP EQP LS 46 Revision A 1 COMORES 5 Z Prism A Prism Polariser gt gt Figure 1 5 DIC prism polariser locations 4 Figure 1 6 The lower DIC Prism and Upper Polariser The electronic version of this document is the latest version It is the responsibility of the individual to ensure that any paper material is the current version Printed material is uncontrolled documentation Last Revised 13 October 2014 Page 12 of 31 Title SG52 Microscopes Department STEP Document No OP EQP LS 46 Revision A 1 Set up K hler illumination in brightfield See section 1 4 1 Place the polariser on the condenser Engage the Polariser in the filter wheel below the objectives Remove an eyepiece and rotate the polariser until the pattern visible in Fig 1 7 is visible Slide the DIC prism under the objective Engage the appropriate DIC prism in the condenser By moving the slider of the prism we can adjust the level of wave retardation NOOR WD Figure 1 7 Desired Polarisation Pattern for DIC 1 5 Microscope care Know your working
13. best used for fluorescence imaging as the filter cubes only work properly in this mode DIC and Phase Contrast are not possible in this mode The lamps for reflection vary with the microscopes They will either be a high power mercury short arc lamp or xenon arc lamp The arc lamps are housed in units called the MT20 These provide very high power illumination required by the Andor and Hamamatsu cameras for high speed imaging 1 3 3 Transmission filters diffusers A diffuser is in place in the lamp housing to prevent direct imaging of the tungsten filament at low magnifications The diffuser may be moved out of the light path when using high magnifications There is the option of using other optical filters for fluorescence techniques which also may be moved in and out of the light path as required 1 3 4 Field Stop This is the first iris diaphragm useful when setting up Kolher illumination 1 3 5 Condenser Assembly and Aperture Stop The condenser assembly creates a concentrated cone of light to illuminate the sample It contains a couple of lenses to do this and a number of components which can be switched in at will either phase rings or DIC prisms or nothing when brightfield illumination is required The condenser also contains an iris diaphragm known as the aperture stop 1 3 6 Stage Put your samples on the stage 1 3 7 Objective Lens Turret This is a rotating turret which will contain a number of objective lenses 1 3 8 Filt
14. ch in a separate display Select the time lapse icon a left click and drag to surround the stage icon Decide on the length of time for your experiment Repeat is the number of times the commands in the time loop frame are to be executed The cycle time is the repetition time and its unit The approximate minimal cycle time is the minimal possible duration of a single cycle So for example Approximate minimal cycle time 642ms therefore you can use anything above 642ms If you decide that the cycle time is 650ms and you want to run your experiment for 5 minutes you will need to repeat the experiment 468 times to achieve total loop duration of 5 min To begin your experiment ensure the live acquisition mode is off If the experiment is brightfield you do not need the burner on If the experiment is fluorescent the burner should be on a minimum of 10 minutes before the experiment and the halogen lamp should be off T4 BY prepare Start Vv And your experiment will automatically run for 5 minutes To pause an experiment select the pause icon Click on verify To analyse the data from your experiment return to your database and select your experiment double click on the image so it appears in the box on the left if you then click on it it will appear fully in your field of view Go to Image Define ROIs and a small dialog box will pop up If you want to analyse your entire field of view select the rectangle and drag to fit you
15. control over the optics Phase Contrast is a technique primarily used to image transparent objects on a transparent substrate It accentuates differences in thickness by using the phenomenon of phase shifts which occur due to the differing path lengths of the light through the sample The greater the optical path length the greater the phase shift which results in darker portions of the image The electronic version of this document is the latest version It is the responsibility of the individual to ensure that any paper material is the current version Printed material is uncontrolled documentation Last Revised 13 October 2014 Page 9 of 31 Title SG52 Microscopes Department STEP Document No OP EQP LS 46 Revision A 1 Phase contrast requires special optical elements to work In the condenser assembly there are a number of phase slits which provide a ring of light In a phase contrast enabled objective there is a phase plate as shown in Figure 1 4 pu F igure 1 4 A phase plate inside an objective lens Setting Up Phase Contrast Imaging 1 Set up Kohler illumination see section 1 4 1 2 Ensure you are using the right phase annulus that corresponds to the objective There are a bunch of these in the condenser simply rotate by hand until you have the one you want That s it You can tell if things are going right when edge features of your sample glow brighter than the background Optimising Phase Contrast Imaging
16. cturing defects but the most likely observation of this will be through imaging of off axis rays Again a stopped down aperture will prevent this Coma Coma produces similar effects to spherical aberration only the distortion is not symmetrical Point sources will be smudgea and have a tail like a comet This is due to variations of magnification of off axis rays Field Curvature Filed Curvature is an instance where the image and or object planes of an element are curved not planar This is manifested by the periphery of an image being out of focus when the centre is focussed and vice versa Corrective elements to flatten the field are easily incorporated in all objectives Distortion Distortion is unavoidable apertures increase distortion and the thicker the lens the greater the distortion Distortion is commonly seen as warping of the image such as barrel and pin cushion effect 5 A pinhole camera with no optical elements except the pinhole aperture itself is distortion free The electronic version of this document is the latest version It is the responsibility of the individual to ensure that any paper material is the current version Printed material is uncontrolled documentation Last Revised 13 October 2014 Page 16 of 31 Title SG52 MICROSCOPES Department STEP Document No OP EQP LS 46 Revision A1 1 7 Objectives Every objective will have some numbers written on the side to let you know the properties
17. e that any paper material is the current version Printed material is uncontrolled documentation Last Revised 13 October 2014 Page 25 of 31 Title SG52 Microscopes Department STEP Document No OP EQP LS 46 Revision A 1 10 For length and area measurements select the Measurements tab or go to Measurements measurements bar to open the measurements button bar Sample features include vertical line and horizontal line Z When the measurements are drawn they can be saved onto your image 2 4 Working with Regions of Interest 2 4 1 Defining the Region of Interest ROls are parts of images that are defined by the user for subsequent analyses The ROls do not become part of the images even if being displayed that is they do not change the data and can be removed at will Open the Define ROIs dialog window with the ROI button or via Image gt ROI Define ROIs Label PI 1 ROH w 2R0t 2 DELIE e Label By default i e if no name is typed into the box the ROIs are labelled ROI lt consecutive number gt ROIS with identical names will likewise be numbered consecutively Labels can be deleted or changed by clicking on the respective commands in the sub dialog that is opened with the arrow adjacent to the Label box e Color ROIs are colored differently by default The color can be changed by selection from the pull down palette prior to drawing To change the color of an existing ROI use the respective
18. eer 13 1 6 SOME HANDY TERMS AND TPPS AL A oeeeeesesesssssssssssssssssssssssssssssssssssssssssssssessesessssssssesessesessssssssssessssee 14 1 6 1 Common Nomenclature NP sccccccnssesssccccsensssssscccceecnssesssceceuecsssesssceceuensssssscccsseassssssecccseecssssssseceees 14 1 6 2 PTI ONS RER D a E A EEEE EEEE EEEE EEE TEENETE 15 1 7 OBI CDN ESS D D D D rr 17 1 7 1 Using an immersion objective G cccccccccccccccccccsscccensssusseseseeenececccacanccecsasseseeeseeeeececececeuscecsasseseseseeeneneeeeacaneoes IS d 18 LI 5032 MICROSCOSS Re oc ee 20 2 BSSICOLYMPEUS SOP ME Ey sorei a aa 21 2 1 BASIC TRANSMISSION IMAGING nana aaann er amenant nan nner ones 2 1 2 2 TIME LAPSENA PERIMENTS ne ee ie nee E 22 2 3 REFLEENON PIROORESCENT IMAGING csccccseccssvcccsccssccnssensscesncensscnsssenscenssenseseassenssensscescenssessscenscensssesecensvens 23 2 4 gy ORKING WITH REGIONS OF INTEREST iccssscenecvncettotepnennete asin dobtinncphetneiesbed tpnen heir anion sobtinnee setnnnled Nek terre bvenien metinncaimewnntsbaes 26 2 4 1 Mea 2 1 Regiono INC OSL aesir aa T E tata teed A S tetes ten nidieies sed eec 26 Od 2 Makine a measurement of a ROI minson tes eee c diese sd tipecasceaseeeuaiesnsatecsstcasatii eee Re ae 28 2 4 3 DOC TOME SUE ACO Re Lane ae ee dd ee ee eee 28 The electronic version of this document is the latest version It is the responsibility of the individual to ensure that any paper material is the current versio
19. ening until the diaphragm image circumscribes the field of view 7 Don t forget to put the barrel back to 0 position e Adjusting the aperture stop when you are set up may improve results e Double check you are using the correct annulus for your objective 1 4 6 Differential Interference Contrast DIC can be considered complimentary to Phase Contrast in that it also utilizes phase information to produce an image The most notable difference between Phase Contrast and DIC is that DIC highlights large gradients of refractive index As far as which is the best to use DIC has a number of traits which are superior to Phase Contrast but you ll just have to try both Photobleaching Avoision Bonus Because DIC and phase contrast use relatively low levels of light compared to brightfield and the burners DIC can also be used to reduce the effects of photobleaching Simply locate the region of interest using DIC and when everything is set up switch to fluorescent imaging Note that both the light sources need to be on and blanked off accordingly Setting Up DIC Imaging DIC is set up using the standard brightfield condenser It can also be done using the dark field condenser but the dark field aperture must be removed and replaced with a polariser we re not going to cover this here though We need to employ the configuration shown in Fig 1 5 with two polariser prism pairs both above and below the sample The Lower Prism and Upper Pol
20. er Wheel The microscopes have slots available for 6 filters Most of the filters are used in reflection mode In transmission mode there is a blank position with no filter for brightfield imaging section 1 4 1 or a DIC prism section 1 4 5 which should be used 1 3 9 Eyepieces If the image is not directed to one of the cameras you should see some sort of image here 1 3 10 Sideports These are used to attach a number of cameras you typically have to select a mirror position to direct the light to and from the sideports The electronic version of this document is the latest version It is the responsibility of the individual to ensure that any paper material is the current version Printed material is uncontrolled documentation Last Revised 13 October 2014 Page 5 of 31 Title SG52 MICROSCOPES Department STEP Document No OP EQPILS 4 Revision a1 1 4 Basic Setup 1 4 1 Power up down procedure Note Manual microscope 1 Power up procedure e Turn on microscope all parts of microscope are on a single power strip Switching on the socket will turn the lamp cameras etc e f measuring fluorescence turn on MT20 using switch on box at far left Note DO NOT turn on the source if you do not intend to use it i e for brightfield measurements e Turn on Computer e Start Cell F software 2 Power down procedure Remove sample from stage Turn off burner in software Return to 4X objective Wait for burner
21. et up on any microscope with minimum hassle afterwards 1 4 4 Darkfield Illumination Note Colour Microscope Only Darkfield imaging utilises scattered light as opposed to reflected absorbed light see 1 4 1 The advantages of dark field imaging are that objects below the diffraction limit can be imaged in fluorescence or sideband excitation and live and unstained samples may be examined However high intensity illumination must be used and setup can be tricky For this reason unstained samples or samples using unquenchable fluorophores are best used Darkfield imaging requires a special condenser assembly The condenser body IX2 DICD also contains a polariser and prism for use with DIC see section 1 4 5 The dark field condenser lens IX2 TLW can be used with or without an immersion fluid which is WATER The working distance is 3 7mm which is ideal for inverted samples Because of the large working distance the surface tension of water is not high enough to maintain a Suitable droplet So an o ring or petri dish to contain the water is a good idea for an inverted sample the 1mm thickness of the slide may be enough to preclude such a measure 7 Also if you have conjunctivitis it is good practice to simply wear glasses safety or corrective to reduce the risk of passing it on Don t forget to check your objective lens to see if it requires oil water or nothing The electronic version of this document is the latest version It i
22. gt That is the one in the condenser Please use both eyepieces it really is less straining over long periods and we shelled out for stereo microscopes so you may as well use them The electronic version of this document is the latest version It is the responsibility of the individual to ensure that any paper material is the current version Printed material is uncontrolled documentation Last Revised 13 October 2014 Page 7 of 31 Title SG52 Microscopes Department STEP Document No OP EQP LS 46 Revision A 1 Eyepiece separation The distance between eyepieces is adjustable as well simply move the eyepieces together or apart to suit the interpupillary distance Again it s a good idea to remember the position for your own ease and comfort Then you can just sit down at any microscope and adjust to suit in seconds Individual eyepiece focus On every microscope one of the eyepieces will have an adjustable focus to account for differences between eyes The adjustable eyepiece will have a graduated scale printed on it The best procedure for this is to 1 Focus your sample with the fixed eyepiece using one eye 2 Rotate the other eyepiece whilst looking through it thus adjusting it to produce the best image This should only need to be done once as the main microscope focus should suffice for all subsequent samples and objectives Power up It s a good idea to remember this position from the scale so you can s
23. h you wish to work with e g Don t forget the rules for operating the burner The electronic version of this document is the latest version It is the responsibility of the individual to ensure that any paper material is the current version Printed material is uncontrolled documentation Last Revised 13 October 2014 Page 24 of 31 Title SG52 Microscopes Department STEP Document No OP EQP LS 46 Revision A 1 Illumination System MT fx Main Swatch Burner ON i OFF Excitation nie D oo al Dy Intensity LOO Te Burner type MT _ARCIXE 150W anon Burner hours Burner Sample illumination 5 To illuminate your sample click on the shutter F Focus on your specimen using the focus wheels of the microscope 6 To view the image on the screen ensure that the ocular button aL is switched to microscope 7 When your image is focused and in view on the screen click on the snapshot tab in the acquisition toolbar and the image will be displayed in the viewport 8 To insert a scale bar go to image scale bar and select draw into overlay Got to save as and save your image to your designated folder in your preferred format jpeg tiff png 9 To change the properties style and size of the bar and the dimension units of the scale bar go to image scale bar scale bar properties The electronic version of this document is the latest version It is the responsibility of the individual to ensur
24. handy terms and tips 1 6 1 Common Nomenclature Numerical Aperture This is a measurement of the light gathering power of an element or system It is directly linked to the resolving power of the system The higher the N A the higher resolution the objective is capable of NA noSinOmax No is the index of the immersing medium this can be oil air or water Omax IS the half angle of the cone of light being collected in the lens In air the N A can t be bigger than 1 hence the use of immersion fluids for high magnification Water or Oil will be written very clearly on the barrel of an objective to indicate it is suitable for use with immersion fluids DO NOT use fluids on objectives unless the lens specifies it If in doubt consult one of the microscope guardians Resolution There are a number of different methods of determining resolution The simplest for microscopes is the Rayleigh criterion which states the minimum distance between two distinguishable radiating points If you can t tell the two points apart they are beyond the resolution of the microscope You can be limited by your detectors and optics but there is a fundamental limit where the objects you are trying to image are smaller than the wavelength of light you are using To get beyond this limit requires dirty tricks using things such as particles with a non zero momentum quantum mechanics money and or lasers It turns out the resolution r can be defined
25. ic version of this document is the latest version It is the responsibility of the individual to ensure that any paper material is the current version Printed material is uncontrolled documentation Last Revised 13 October 2014 Page 3 of 31 Title SG52 MICROSCOPES Department STEP 1 3 Anatomy of a microscope Figure 1 1 A Microscope The microscopes in SG52 are inverted microscopes so these will be the kind discussed here Figure 1 1 shows a schematic of an inverted microscope 1 Transmission Light Source 2 Reflection Light Source 3 Transmission filters diffusers 4 Field Stop 5 Condenser Assembly and Aperture Stop 6 Stage 7 Objective Lens Turret 8 Filter Wheel 9 Eyepiece 10 Sideport Page 4 of 31 Title SG52 Microscopes Department STEP Document No OP EQP LS 46 Revision A 1 1 3 1 Transmission Light Source Halogen All the microscopes have 2 lamps One for transmission one for reflection Transmission mode allows modes such as Phase Contrast or DIC Usually it is white light from a tungsten filament You will need to buy a filter to insert in the beam path if you want to use a particular wavelength Using a filter cube does not make any sense here so be sure to set the filter wheel so no filter is present 1 3 2 Reflection Light Source Arc Burner In reflection mode the light passes through the objective reflects off the sample and goes back through the objective This mode is
26. lve and disperse and give you an unwanted fluorescence background Then fingerprint grease will be all over the objective and then dust adheres better to the grease and UGH A small drop of the immersion fluid is placed on the lens Then the sample is placed on the stage and the objective is raised using the focus wheel You should be able to see when the fluid drop comes into contact with the sample The sample can then be brought into focus Please clean the lens bottle and any other contaminated surfaces when you are finished Using a small sheet of lens tissue place it on the lens and drag it towards you Let the weight of the tissue do the work and soak up the fluid For water objectives using de ionised water is good practice as this reduces the buildup of mineral deposits on the lens 1 8 Filters Here we aim to list the filters and give a short explanation of how they work Filters remove unwanted wavelengths of light There are 3 basic types Short Pass Long Pass and Band Pass Long Pass allows long wavelengths to pass through but will block short wavelengths Short Pass is the opposite Band Pass will allow a particular group of wavelengths and block anything outside that band The filter s specifications should be written on the mounting Short Pass will be noted as SP or KP It will have the wavelength in nanometers at which e Long Pass will be noted as LP 1g Yes small The drop of immersion fluid should be small
27. n Printed material is uncontrolled documentation Last Revised 13 October 2014 Page 2 of 31 Title SG52 Microscopes Department STEP Document No OP EQP LS 46 Revision A 1 1 THE BASICS 1 1 Ground rules e If in doubt about any of this consult one of the people on listed on the front page We d be delighted to help e DON T change any components on the microscopes without consulting the relevant staff e DON T attempt to clean any optics unless you have been trained e Please use both the log books and booking system http ncsrbooking dcu ie booking e Beconsiderate to other users e There s plenty of information here http www olympusmicro com primer techniques 1 2 MT20 burner operation e The burners for the MT20 cost a lot of money Not following the rules will be punishable by having to buy a new burner e DO NOT forget to switch the burner off at the end of the day e DO NOT run the burner for short times Plan your experiment If somebody is using the microscope after you contact them and see if they want it left on e f the burner has been switched off allow 30 mins to cool before switching back on e Please log any burner related errors or messages e Please ensure theMT20 exhaust is not obstructed e g curtains paper walls to avoid overheating The issue here is the extreme change in temperatures This shortens burner lifetime Again we are avoiding thermal cycling The electron
28. occussed image of Field Stop 5 Adjust the height of the condenser until you can see a clear image of the Field Stop through the eyepieces like In Fig 1 2 Note This might not be possible at high magnifications so use a lower mag to get the condenser and Aperture Stop image into position 6 Open up the field stop do you can just see the edges in the field of view Centre the image of the Field Stop using the two screws at the front of the condenser 7 Open up the Field Stop just until you can no longer see the edges of it in the field of view If you open it all the way you ll be getting a lot of scattered light this won t give you a great image 8 Adjust the Aperture Stop for best effect e For oil immersion the Aperture Stop should be open all the way e Inair closing the Aperture Stop improves contrast but also highlights diffraction artefacts So it s best to try and find the middle ground 1 4 3 Eyepieces The eyepieces provide a final magnification and an image plane on which it is easy for the eye to focus They can be used with and without glasses The best viewing position is a cm or two away What you should be seeing if everything is correctly adjusted is a single round image that is comfortable to look at The diffuser is frosted glass on top of the microscope next to the light source which can be easily swung in and out of the beam That is the variable aperture at the top of the microscope Number 3
29. our camera and a monochromator coupled to a monochromatic camera for selectable emission imaging The DP7 is a 1 5 Megapixel colour camera with a single CCD and exposure time range from 1 44 000 sec to 60 sec Page 20 of 31 Title SG52 MICROSCOPES Department STEP Document No OP EQP LS 46 Revision A1 2 BASIC OLYMPUS SOFTWARE 2 1 Basic Transmission Imaging 1 Inthe cell software open create your database 2 Click on the microscope control icon 3 to open up the dialog box Microscope M a 20086 5 um Change limits vw a Objectives UPLSAPO 60x v Fluorescence turret Le DaFiTR v CC am a 3 Select the objective of choice Gel once selected the nosepiece moves automatically into the selected position 4 Ensure that the lamp is turned on from A to p Use the slider to increase decrease the intensity of the transmission light Focus on your specimen using the focus wheels of the microscope Page 21 of 31 Title SG52 Microscopes Department STEP Document No OP EQP LS 46 Revision A 1 5 To view the image on the screen ensure that the ocular button ay is switched to camera 6 Focus the image and set up proper illumination see section 1 4 1 id 7 Click on the snapshot button in the acquisition toolbar and the image will be displayed in the viewport 8 To insert a scale bar go to image scale bar and select draw into overlay Go to save as and save
30. r field of view You can label the rectangle Close the dialog box Go to Measure Kinetics select your labelled rectangle in available ROIs and click OK Minimise the field of view and behind it a spreadsheet with your data will appear You can save this as an excel file to your folder Another nice feature is that a graph is automatically generated so you can see your data quickly without plotting it 2 3 Reflection Fluorescent Imaging 1 2 In the Cell R software open create your database Click on the microscope control icon 3 to open up the dialog box from here you can select the objective which you want to use and ensure that the correct Filter and Condenser Free or BF are selected The electronic version of this document is the latest version It is the responsibility of the individual to ensure that any paper material is the current version Printed material is uncontrolled documentation Last Revised 13 October 2014 Page 23 of 31 Title SG52 Microscopes Department STEP Document No OP EQP LS 46 Revision A 1 Microscope E Ed a Him Change lirmiks Objectives UPLSAPO 10 tat Fluorescence turret M DAFITR LA Condenser Fres a Lamp sr l OC 3 Focus on the correct focal plane for you sample Tag a6 4 Click on the Illumination Settings icon ie to open up the dialogue box Switch the burner to the on position FITC and select the excitation filter whic
31. re light from 600nm will be transmitted with a peak transmission at 625nm The electronic version of this document is the latest version It is the responsibility of the individual to ensure that any paper material is the current version Printed material is uncontrolled documentation Last Revised 13 October 2014 Page 19 of 31 Title SG52 MICROSCOPES Department STEP 1 9 SG52 Microscopes There are 4 microscopes with different capabilities The manual microscope is the only one capable of phase contrast imaging The other 3 are capable of DIC All can do brightfield and one can do brightfield and darkfield ANDOR HAMAMATSU MANUAL COLOUR Phase Contrast Z SSS oS S A S O Brightfield ee ee S ee O a O Darkfield oo Monochromator y O MT20 CY A a O rl Speciality High Sensitivity Hi res amp High DR Quick amp Easy It is in colour Spectrometer Andor v The Andor camera is highly sensitive Andor claims single photon sensitive It is a backside illuminated 512x512 camera with a QE of 90 and a max readout rate of 10MHz It s quite a machine A TIRF stage has been designed for use with this microscope Hamamatsu The Hamamatsu is a high resolution 1344x1024 high dynamic range camera Great for imaging fluorescent dyes over long exposures Manual A colour TV camera and a colour Firewire camera make this microscope the best for a quick check of you sample Colour This features an Olympus DP71 col
32. s or Z stack as long as it has the same number of frames respectively layers as the data set of interest e Dimensions tab In the fields Color Channels Z Layers and Time Frames you can choose the range of images in the different dimensions for which the analysis shall be performed in case only a subset of the data is of interest By default the entire data set is selected e Frames You can select every second third fourth image and so on to be analysed Copy non processed data If this option is NOT selected the newly created data set will contain only the selected subset of the source data If the option is selected a copy of the original Source data will be created in which the selected subset range Is processed background subtracted while the rest is identical to the source data The electronic version of this document is the latest version It is the responsibility of the individual to ensure that any paper material is the current version Printed material is uncontrolled documentation Last Revised 13 October 2014 Page 29 of 31 Title SG52 Microscopes Department STEP Document No OP EQP LS 46 Revision A 1 Background Background Settings Dimenciarr Colo Chances Pus 4 Fura lerers Figure 2 2 The background Dimension window The electronic version of this document is the latest version It is the responsibility of the individual to ensure that any paper material is the current version Printed material
33. s the responsibility of the individual to ensure that any paper material is the current version Printed material is uncontrolled documentation Last Revised 13 October 2014 Page 8 of 31 Title SG52 Microscopes BSNL ie A DE rs Er Figure 1 3 the same sample imaged in Brightfield Left and Darkfield Right illumination 1 Engage the darkfield condenser by tilting back the support column Loosen the grub screw on the side of the support column Swing the condenser into place Tighten the grub screw whilst rocking the condenser slightly side to side to properly locate the condenser 2 Focus the sample and obtain K hler Illumination This can be tricky as the aperture won t be imaged clearly 3 Temporarily remove an eyepiece and observe the darkfield light disc and stop 4 Use the stage centring screws to centre the DF condenser so that the bright disk is completely covered by the central stop 5 Replace the eyepiece 6 Adjust the barrel of the DF condenser until a decent dark field image is obtained The condenser lens will be quite close to the sample So be careful you don t cause a crash 7 Fiddle around hits the settings a bit to try and get a nice image 1 4 5 Phase Contrast Note Colour Microscope Only When you setup the optics to use phase contrast the technique is the same as for brightfield imaging mentioned above However phase contrast is set up only on the manual microscope so you won t have software
34. t to visualise the experiment as it runs Automap is selected and image type colour which relates to the colour of your filter Standard Commands Image Acquisition Z stack Multicolour Time Lapse PNG is a nice new modern standard that can produce better quality images than JPEG PNG and TIFF images are not lossy TIFF images can be big If you really want a small file size use JPG but detail will be lost The electronic version of this document is the latest version It is the responsibility of the individual to ensure that any paper material is the current version Printed material is uncontrolled documentation Last Revised 13 October 2014 Page 22 of 31 Title SG52 Microscopes Department STEP 10 11 12 Document No OP EQP LS 46 Revision A 1 To monitor a position in your sample over time you need to create a position using the stage function RAA Select the icon Ex on the main toolbar and a dialog window will open up Simply focus your sample in the pull down menu select New List and type in a name for your position and click Add and Save the position You can save more than 1 position under one name RAA In experiment manager select the stage icon aS left click and drag to surround the image acquisition icon in the experiment plan Select the position you have saved from the pull down menu and select all positions in one display if you have 1 position if you have 2 positions you can see ea
35. te This function will create a new data set The original source data remain untouched The electronic version of this document is the latest version It is the responsibility of the individual to ensure that any paper material is the current version Printed material is uncontrolled documentation Last Revised 13 October 2014 Page 28 of 31 Title SG52 Microscopes Revision At Hackground NELCYETESETESTOTTEST Background Settings Danensions Backhoe Sutractor 3 achko ound Subtraction cCorstare O constant Saect ROES from Image 2 Pura 3 POs OR Select HOt J Image ROI 1 ROI Seckoround Subtraction Constant Select Bachg ound baage ROI 3 Fura v Figure 2 1 The background window Background Settings tab e Constant This option subtracts a fixed value from all pixel intensities in all images of all channels The constant has to be typed into the box or can be changed by using the adjacent scroll arrows e ROI The most useful way is to mark a ROI in an image area that contains only background intensity and use the average intensity in this ROI as background value This value is determined individually in each image of each channel e Image This option allows subtracting a predetermined background image set from each of the images of the data set The background image has to have the same X Y size and the same number of channels as the data set to be corrected It can be either a single image or a time serie
36. to cool Close down Cell F software Shut down computer Turn off MT20 Note MT20 MUST be left switched on for the fans to cool the lamp for at least 10 min after being switched off The Burner should not be switched back on for 30 mins Frequently switching a burner on and off reduces its lifetime If the burner will definitely be used again by you or someone else within a few hours burner should be left on and the next person to use it should be notified 1 4 2 Kohler Illumination Brightfield Kolher illumination only has to be setup for transmission imaging There is an optimum illumination setup to achieve the best results This makes sure you don t image the actual light source and gain the most even illumination with a minimum of unwanted scattering This should be used for both reflection and transmission You don t have to worry about it so much in reflection so we ll only be covering the transmission setup The condenser provides even illumination to the sample The goal is to set up an image of the field stop in the same focal plane as your sample 1 The condenser wheel should be set to the brightfield position and you should be getting some light onto the sample 2 Focus your sample roughly Page 6 of 31 Title SG52 Microscopes Department STEP Document No OP EQP LS 46 Revision A 1 3 If you are using 4x objectives use the diffuser 4 Close the Field Stop down all the way Figure 1 2 F
37. too much and it will dribble down the sides of the objective and gunk up the optics and jam the objective into the turret 1 KP Kurz Pass which is German for Short Pass The electronic version of this document is the latest version It is the responsibility of the individual to ensure that any paper material is the current version Printed material is uncontrolled documentation Last Revised 13 October 2014 Page 18 of 31 Title SG52 Microscopes Department STEP Document No OP EQP LS 46 Revision A 1 e Band Pass specifications are usually written as something like 625 25 meaning it allows wavelengths through 25nm either side of 625nm Neutral Density Filters are not wavelength selective and are used to attenuate light from strong sources Diffusion Filters are used to scatter light and are most commonly used to provide even illumination and prevent imaging of the light source at low magnification The typical fluorescence microscopy filter consists of 3 components shown in Fig 1 11 known as a filter cube This cube creates 2 different light paths The excitation light path and the emission light path Typically the emission light path is a narrow band of light and therefore may contain very little power for this reason care has to be taken with sample illumination Sample Excitation Filter Dichroic Mirror Emission Filter Emission Filter Figure 1 11 Filter Cube Schematic T8 That is a window whe
38. w the shape can be adjusted again As always a right mouse click terminates the command Q Rectangle A Rectangle opens the size and shape of which can be adjusted by dragging with the left mouse button o Rotated rectangle With this command rectangles of arbitrary orientation can be drawn The left mouse button toggles between the Move and the Rotate modes indicated by the crosshair and the circular arrow respectively in one corner Dragging without a mouse button clicked performs the moving and rotating In either mode a left button drag changes size and shape The best way to draw is to open a rectangle at any position and rotate it so that the edges are parallel to the desired position Then change to Move mode and position the corner opposite to the crosshair to its final position and finally adjust the size of the rectangle via left button mouse drag Move This enables to move any ROI highlighted in the Active ROI list x Delete This removes the ROI highlighted in the Active ROI list The electronic version of this document is the latest version It is the responsibility of the individual to ensure that any paper material is the current version Printed material is uncontrolled documentation Last Revised 13 October 2014 Page 27 of 31 Title SG52 Microscopes Department STEP Document No OP EQP LS 46 Revision A 1 2 4 2 Making a measurement of a ROI If the ROIS are already drawn when any of
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