lab 6 confocal answers

lab 6 confocal answers - 1. Why can the confocal microscope...

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Unformatted text preview: 1. Why can the confocal microscope obtain optical sectioning of samples? Siwclrnuil “bit-"h H “mm "l‘i‘iTi-‘T V i . I .33th cx'cilulinn " I Fuhl ‘ The confocal microscope incorporates the ideas of point—by—point illumination of the specimen and rejection of out-of—focus light. in a confocal system there are two pinholes: A illumination pinhole for point light source and a detecting pinhole to get rid of out-of—focus image. Thus there are two point spread function: PSFs for source light which describes the distribution of point light in specimen plane and PSFd for detecting light which describes the distribution of point light image on the detecting plane. Since they two are independent events, mathematically, the probability of two independent events is the product of that two probabilities. So, the total PSF of the confocal system is defined as PSFcf = PSFs x PSFd. Since probability is a always smaller than 1, the product of two probability is always smaller than its individual probability. For out-of—focus image, the farther a point is off the focal plane, the more dramatic the signal decreases. It gets so weak at some distance that it is well below the detecting threshold of the system then is no longer detectable. This is the fundamental mechanism of confocal effect or optical sectioning. Light from specimen parts in focus can pass through the tiny aperture and reach the detector, whereas light from out—of—focus specimen parts is mostly blocked. The effect of this is that only light from a very thin specimen layer is detected. This property of confocal microscopy is called "optical sectioning,“ and opens the possibility to study very thin (less than 1 pm) sections of thick specimens. By recording a number of such optical sections, refocusing the microscope slightly between individual sections, it is possible to record a "stack" of images representing the three—dimensional specimen structure. 2. Why does this con focal microscope use a scanning laser spot instead ofo con'.:e.ntfionr.nlr light source? Traditional widefield epi-fluoresccncc microscope objectives focus a wide cone of illumination over a large volume of the specimen, which is uniformly and simultaneously illuminated. A majority of the fluorescence emission is directed back towards the microscope is gathered by the objective and projected into the cycpieces or detector. The result is a significant amount of signal due to emitted background light and autofluorescence originating from areas above and below the focal plane, which seriously reduces resolution and image contrast. The laser illumination source in confocal microscopy is first expanded to fill the objective rear aperture, and then focused by the lens system to a very small spot at the focal plane. The size of the illumination point ranges from approximately 0.25 pm to 0.8 pm in diameter, depending on the objective numerical aperture, and 0.5 pm to 1.5 pm deep at the brightest intensity. In laser scanning confocal microscopy, the image of an extended specimen is generated by scanning the focused beam across a defined area in a raster pattern controlled by two high-speed oscillating mirrors driven with galvanometer motors. As each scan line passes along the specimen in the lateral focal plane, fluorescence emission is collected by the objective and passed back through the confocal optical system. After leaving the scanning mirrors, the fluorescence emission passes directly through the dichromatic mirror and is focused at the detector pinhole aperture. Fluorescence emission remains in a steady position at the pinhole aperture, but fluctuates with respect to intensity over time as the illumination spot traverses the specimen producing variations in excitation. Through this method, much of the autofluoresccnee from areas above and below the focal plane do not affect the image. Laser scanning allows for very precise regulation of wavelength and excitation intensity. It also produces images that have far more detail than images produced with microscopes using a conventional light source. It I L .Sfiwpie 1%. of . 4mg «.1 i gala-j? F F" a 1 fl "79 4’13 .3 a“! ’0 1mm: 4. \%W23¢€&LJL 8/ I03 imam“? m ml Eamr/ m We 003/7; mm a 2m an M a mimosa: a. you a/fl a £3 #51,; .4qu 7w 3% WK 5;}, a; fax or? L w// My #ng A) u/mz ,_.. J 055.5 Wye) :79? mm A, Jc it. wad) an ode w} ‘2” 0g}! at) cal/er fax/Z £3 7v: MI mm @201 L1? m, (430% {M w 23. dug, (a 5m 0434 BM 1%; .m/s J m (an. ’3 f 4) The theoretical limit of resolution is 139.4 nm in the x-y direction, and 235.8 nm in the z direction. In the picture on the previous page, we show detailed measurements about the parameters of an actin filament bundle. According to the above data, we were able to clearly resolve the bundle with a width of about 600 nm. It appears cw that can be resolved in the picture, such as smaller actin bundles and mitochondria; however, we did not perform measurements on these structures. Therefore, it seems safe to conclude that we are clearly within an order of magnitude of the theoretical limit of resolution, and we come fairly close to the limit, but we do not really approach it. The resolution was increased by scanning with higher pixel numbers and scan rate. 5) The thickness we found to be roughly 3.4 Ilni when we were doing the sectioning;r scan since we used 1'? slices and a 200 nm step size. From the post processed 3-d image that we created, a rough estimate of the thickness of the cell against the scale provided seems to give a thickness of about ~2 um. Since the cell is an elial cell, it is found on the outer lining of the artery so having a fairly thin cell aidyn the diffusion and filtering of water and oxygen to the blood cells. a 6. Discuss some practical advantages of the confocal microscope over conventional fluorescent microscopy for imaging live cells. What data would be uniquely accessible with the confocal microscope? if we do not care about the spatial distribution or tomograpghy, do not need a 3-D construction, have no problem with thick specimen or SNR, we can just use conventional fluorescence microscope to get the image easily. However, there are several problematic situations in fluorescence microscopy that we need to use confocal microscopy. In digital camera based fluorescece microscopy, multi-labeling is handled sequentially by change filterset in turn and take separate shot then display them in different channel with or without an overlay. Time delay in several seconds between channels is inevitable. If the time delay is intolerable in your application, confocal is your only choice. in confocal system with maximum number of PMTs installed, up to six detecting channels can operate simultaneously without any time delay. So it is ideal for Living cell imaging of multi-labeled marker for highly dynamic events. Background fluorescence is sometimes the headache of fluorescence microscopy. It makes picture looking bad. buries weak signal in background and reduce general contrast thus redUCe obtainable resolution. This problem is more severe in tissue section and thick specimen than in cultured cell specimen. In digital camera based system, the common way to overcome it is simply lessen the excitation intensity or reduce the exposure time. In confocal system, most of the out-of-focal-plane signal is restricted by pinhole resulting in a cleaner background and detected background can be further manipulated by play with parameter on PMTs, i.e. reducing gain and raising 93‘!th added on the PMTs, or averaging__s__ignal on the detected frames by digital image processing algorithm. It must m out; the assumption here is you have a general strong signal that can survive the discard of most of the original signal due to the confocal effects. Also, if you need to get a 3-D image reconstruction of your living cell or spatial distribution of the labeling or tomography of the cell structure has significant impact on your study, you can only choose confocal microscope. However, the imaging of living specimens adds the challenge of maintaining the life and normal function of the organism. There are some difficulties involved in preparing the sample for viewing as is the case in conventional microscopy. But on top of that the effect of photo damage (bleaching) on the specimen caused by the focused high intensity excitation light must be taken into account/r This is compounded by the repeated exposure required for tracking the cellular dynamics—a problem that is worsened for SD data collection. Fluorescence also introduces the problem of the fluorophore influencing the cell behavior. ...
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lab 6 confocal answers - 1. Why can the confocal microscope...

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