Fluorescence Microscopy Submitted by Maheep Singh (0905569) Abstract This experiment qualitatively describes the three microscopy techniques

that are widely used to analyse biological samples. This report analyses the advantages and disadvantages of different techniques and the method used to achieve images. It was concluded that STED microscopy gives rise to the finest images whereas confocal technique holds a potential of better imaging than widefield technique. Abbey limits using different dyes are calculated and the advantages and disadvantages of STED are also closely studied. 1. Introduction Fluorescence microscopy is the most widely used imaging tool in biology. This originates from the non-invasive characteristics of optical microscopy which allow the probing of structures and functions of live cells in the 3-dimensional space at the submicron scale. Moreover, fluorescence microscopy provides high sensitivity down to the single-molecule level which permits the observation of molecules and organelle-specific signals. However, the resolution power of far-field light microscopy is constrained by the diffraction limit of light, as explained by Ernst Abbe more than a century ago: any object, no matter how small, will be imaged by a conventional optical system as a finite-sized spot, with a minimum dimension obtained for point-like objects (such as single molecules) approximately equal to half the wavelength of the incident light beam. This experiment was conducted with an aim to understand how different fluorescent microscopy techniques work and the different advantages and disadvantages they bore. The three techniques studied in the experiment are Widefield microscopy, confocal microscopy and STED (Stimulated Emission Depletion) 1.1 Widefield Microscopy This microscopy is one of the most basic techniques which can be found in any standard biological and medical-research based laboratory. Widefield microscopy is the starting point in the career of any microscopist. A widefield microscope can either be inverted or upright depending on the experiment. A halogen lamp is used to illuminate the sample. The resolution achieved through this kind of microscopy is lower than that attained through Confocal microscopy and STED. [1] With the use of widefield scanning, an area of interest is illuminated with light and an image is recorded with the aid of a CCD camera. Widefield Microscopy results in capturing of both in-focus and out-of-focus light. 1.2 Confocal Microscopy This microscopy generates slightly better resolved images than widefield microscopy. Its discovery follows after extensive usage of conventional microscopes undergoing Widefield microscopy. Confocal Microscopy produces a significant imaging improvement and allows the collection of data in 3D as well. It focuses a beam down and collects all of the emitted light using a photomultiplier tube. As opposed to Widefield microscopy, it scans the beam across the sample due to which the resultant resolution is higher. This process also undergoes

sectioning the removal of out-of-focus light with the aid of a light pinhole. It doesnt use a lamp but uses a laser source for illumination and fluorescence instead. [4] 1.3 STED Also known as Super-resolution microscopy because of the high resolution it offers in the images, STED is one of the finest discoveries in the world of microscopy. It uses non-linear de-excitation of fluorescent dyes to overcome the Abbe limit imposed by diffraction with standard confocal laser scanning microscopies. STED has a potential of achieving unlimited resolution independent of the wavelength of light. It relies on the concept of fluorescent depletion in which a laser first excites and fluoresces the targeted fluorophore molecule and then uses a depletion beam to cancel all the unwanted fluorescence. Hence STED has an ability to cause fluorescence at a specific area. 2. Methods 2.1 Widefield Microscopy 2.1.1 Sample Preparation First of all the sample is prepared. Due to the short-notice nature of the experiment, the sample was only analogous to the real conditions. A piece of paper was used as an equivalent to tissue and a green highlighter was used as a dye. Half of the experimentalists carried the experiment with their sample (lens paper and green highlighter) embedded in oil while others carried theirs in air. A coverslip was then applied over the sample and the sides were shut properly by using nail polish. It was made sure that the oil was dropped directly over the area of the specimen to be examined. The set up was allowed to dry for a further 10 minutes. It is important to close the slide using a nail polish or anything equivalent in order to seal the cell culture off completely from oxygen and the outside environment. This will enable the specimen to remain as it is without being deteriorated. The sealing of the slide is important to avoid air bubbles which can be quite a problem for new microscopists. Bubbles can cause optical artefacts at the place where the air meets the water. If the bubble is large enough, then it can also cause the resolution to be lower. The reason for using a drop of immersion oil was to enable a higher resolution of the image obtained from the microscopy. The oil drop acts as a bridge between the glass slide and the glass in the lens. This helps to optimise the light path from the microscope and allows a higher resolution. Oil has a refractive index equivalent to that of the glass, therefore it helps in avoiding scattering of light via refraction and reflection from glass to oil or vice-versa (as bending of light is avoided because both the interfaces have same refractive index). Oil was also applied on the slide so that the objective is in direct contact with the drop. Therefore, the oil-embedded specimen results in a better resolution as light scattering is absent due to which there is negligible spherical aberration [2] Embedding medium such as air, water or PBS (Sodium perborate) were avoided as their refractive index were

totally different from that of glass used in objective and the slides; resulting in a potential refractive mis-match. The thickness of the coverslip is also quite an important parameter in observing images of higher resolutions as thicker coverslips can result in the production of inferior images. This is due the fact that thicker interfaces encourage higher scattering which leads to more refraction and resulting in greater optical aberration.

2.1.2

Widefield Imaging An optical tissue was used to clean the objective. An oil drop was placed on the sample plate which was placed on the holder. The halogen lamp was turned on through a switch present on the base of the microscope. The sample was slowly brought towards the objective and the image focus was closely monitored through the eyepiece. There are a set of defined conditions for the optimal microscopy (resolution and contrast) of the illuminated specimen known as Kohler illumination. These conditions are very necessary for brightfield imaging. The five steps for achieving Kohler illumination are as follows: a) The sample was brought to focus initially by using the focussing knobs provided on the side of the microscope. b) The field diaphragm was then closed so that just the focussed picture can be looked at. c) Then the edge of the diaphragm was focussed by adjusting the condenser height. This is done by the knobs which raise or lower the entire condenser. d) Fourth step is to get the focussed image on the centre of the picture seen through the oculars. This is done by altering the centring screws available on the Substage condenser. e) Finally the field diaphragm, which was closed in step b) in order to get rid of out of unnecessary shadows visible initially, was opened until it is at the edge of the field of view. [3] f) The condenser aperture diaphragm, which is located in the condenser unit, can be focussed up and down. Hence the Kohler illumination helps to optimise the field of view obtained from Widefield microscopy. Failure to use the Kohler illumination can result in a poor resolution of the images and some uneven lit pictures. Blurred images can also be seen if this illumination is not met. Once the sample is mounted and focussed on the sample plane, it is fine focussed by using the relavant knobs. The sample was then placed in the middle of the field of view by using stage control. The difference between the oil and air embedded samples were observed. The embedded sample was seen with a better resolution as compared to the air embedded sample. The reason for this is discussed above already.

There is a direct link between the refractive index n of the immersion medium and the numerical aperture (NA): (1) So a higher n results in a bigger NA resulting in a lower Abbe Limit d: (2) The advantage of oil embedded sample is that it undergoes a minimum amount of scattering of light and hence the shape of the object and the image are roughly the same. The resolution of the obtained image is relatively higher too. The only possible disadvantage of using an embedded sample is that the sample may lose its original properties or may appear differently due to the different colour of the embedding sample. Also, it may be difficult to visualise the various parts of the field of view after the introduction of the embedding medium. But generally the advantages of using embedded 2.2 Confocal and STED Microscopy 2.2.1 Confocal Microscopy This was achieved through another piece of hi-tech equipment which could be tuned between Confocal and STED simultaneously. There are various advantages of using Confocal microscopy over the conventional widefield fluorescence microscopy such as: a) Confocal microscopy offers the ability to control depth of field, elimination or reduction of background information away from the focal plane. b) Confocal microscopy also involves sectioning and gets rid of unwanted out-of-focus information which widefield microscopy shows. c) Confocal microscopy can be used in specimen whose thickness exceeds the dimensions of the focal plane. To achieve confocal microscopy, cardial myocite sample emedded in PVA (an alcohol) was mounted in the same as the lens paper was mounted for widefield microscopy. The wavelength of the laser was matched with the excitation wavelength of the dye used. After this the scan button was pressed and after some time, an image was obtained on the screen attached to the apparatus. A Fluorophore is a component of a molecule which causes that molecule to fluoresce. Its emission wavelength is greater than the exiting wavelength. Fluorophores used in Confocal microscopy are i) ii) A cyanide2 dye cy2- whose excitation wavelength is 488nm and emission wavelength is 510 nm An atto 647 dye whose excitation wavelength is 635nm and emission is 647nm

2.2.2

STED Microscopy A similar procedure was employed for STED as the apparatus used was the same. Only atto-647 dye was used in this experiment. A second depleting laser beam was made use in

order to cancel the fluorescence of the undesired region from the sample. The depletion wavelength was set to 750nm and excitation wavelength varied with the fluorophore used.

Results Figure 1 and 2 summarises the results obtained from widefield and confocal microscopy

Figure 2: Image obtained throug h widefield microscopy using cy2

Figure 1: Image obtained throug h confocal microscopy using cy2

Figure 3 shows an image obtained from STED while fig. 4 presents a comparison with its Confocal counterpart on the right

Figure 3: image obtained through STED

Figure 4: image obtained through confocal atto-647

The Abbe limit for Confocal experiment is calculated from Eq1 and Eq2 defined earlier.

NA of the objective is 1.4 and is provided prior to the experiment. (effective wavelength) was used instead of wavelength) (excitation wavelength) or (emission

= 89.1 nm (cy2 experiment)

= 114.5 nm (for ato 647 experiment)

Discussion It can be clearly seen from the two images (Figure 1 and 2), that the resolution obtained through confocal microscopy is much higher than its widefield counterpart. Also the sectioning of out-offocus regions can be clearly visualised. The brightness of the image is reduced in confocal microscopy which is at the expense of a better contrast, allowing the viewer to visualise better. Also confocal microscopy produces a much sharper image The similarities between figure 1 and 2 is that both the images use darkfield microscopy. Figure 3 and 4 compares the images obtained through STED and Confocal technique using the same fluorophore. It can be clearly seen that the STED experiment results in a better resolved image. The contrast visible is higher than that of the Confocal experiment. This is due to the fact that STED shows a very high resolution by avoiding diffraction. Abbe limit for STED is not calculated as the technique has the ability to go below the abbey limit in order to produce a high resolution. Another important discussion is about the advantages and disadvantages of using STED microscopy. STED has numerous advantages over Confocal and the other conventional microscopy techniques, such as high resolution, highly computerised. Another promising advantage of STED is that it can record living cells at a much higher resolution. [5] Although STED looks better than most other techniques, it does have a few drawbacks. One being, that the machinery used is quite complicated and requires a lot of training to be used. Also, the rate of image acquisition for larger field of views is slow as it needs to scan the sample throughout to retrieve an image. Also, the resolution along the optical axis is still fairly limited. STED can also lead to sever photo-bleaching of the sample as it requires a large number of activation-localisation-bleaching cycles [6] As far as the comparison of the three techniques is concerned, STED is generally the one that is preferred. However, because STED apparatus costs a lot and cannot be afforded by every laboratory, Confocal technique is also often used. Since STED requires a lot of prior knowledge about the fluorophore and the exact wavelength to be used to excite/deplete the sample, it is often not used. STED gives rise to a lot of phototoxicity, in which case Confocal or widefield microscopy are used. If it is a large field of view that is to be investigated, then Confocal microscopy is preferred to STED.

STED is generally used when highest amount of resolution is to be required to study some of the minute features of a cell culture. Conclusion Confocal microscopy eliminates the various out of focus spots visible on widefield microscopy (figure 1 and 2). STED shows a much superior image than the Confocal as its abbey limit goes beyond the diffraction limit. The Abbe limit achieved through cy2 fluorophore was lower than the ato 647 experiment. It can also be concluded that STED is one of the superior techniques available in microscopy today. It provides a significantly high resolution such that that diffraction is nearly diminished. References 1. 2. Greg Martin(2005),Widefield Microscopies Available at the Keck Center, Available at: http://depts.washington.edu/keck/Widefield/equipment.htm (accessed: 20/12/2011) David B. Frankhauser (2004) , Immersion Oil Microscopy, Available at: http://biology.clc.uc.edu/fankhauser/labs/microscope/Oil_Immersion.htm (accessed 18/12/2011) http://www.einstein.yu.edu/aif/instructions/koehler/koehler.htm (accesed 19/12/2011) D. Semwogerere, E.R. Weeks, Confocal Microscopy, (date and journal not known) Volker Westphal, Silvio O. Rizzoli, Marcel A. Lauterbach, Dirk Kamin, Reinhard Jahn, Stefan W. Hell (2008). "Video-Rate Far-FieldOptical Nanoscopy Dissects Synaptic Vesicle Movement". Science 320: 246249 P. Dedecker, J. Hofkens, J. Hotta, Diffraction-unlimited optical microscopy, Materials Today, vol.11, pages 12-21, 2008

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