Epi-illumination microscope

These small molecule probes of DNA (ds and ss) and RNA do not identify organisms, but indicate probable viability because the genome is intact. When cells in suspension or on a slide are stained and examined under a UV-epi-illuminated microscope or in a flow cytometer, with or without cell sorting, then viability can be estimated with fairly high precision and accuracy. This and related topics are developed in Ref. and in portions of the molecular probes pages (www.probes.com), which the reader may find particularly useful. 

Voor and co-workers constructed a Raman microprobe around an existing microscope and Raman spectrograph [5]. The system is suitable for undergraduate research and is also used in advanced materials science instruction. The system is assembled from a small argon ion laser and an epi-illumination microscope designed for materials inspection. The instrument design, shown in Fig, 1, is straightforward and could be built by most chemistry or physics faculty members. Instructors who intend to build their own Raman microprobes should remember to remove or to disable and cover the binocular eyepieces that are components of most microscopes. 

In a microscope, standard polarized epi-illumination cannot distinguish order from disorder in the polar direction (defined as the optical axis) because epi-illumination is polarized transverse to the optical axis and observation is along the optical axis at 180°. However, microscope TIR illumination can be partially polarized in the optical axis direction (the z-direction of Section 7.2) and can thereby detect order in the polar angle direction. Timbs and Thompson(102) used this feature to confirm that the popular lipid probe 3,3 -dioctadecylindocarbocyanine (dil) resides in a supported lipid monolayer with its dipoles parallel to the membrane surface, but labeled antibodies bound to the membrane exhibit totally random orientations. 

TIRF is an experimentally simple technique for selective excitation of fluorophores on or near a surface. It can be set up on a standard upright or inverted microscope, preferably but not necessarily with a laser source, or in a nonmicroscopic custom setup or commercial spectrofluorimeter. In a microscope, the TIRF setup is compatible and rapidly interchangeable with bright-field, dark-field, phase contrast, and epi-illumination and accommodates a wide variety of common microscope objectives without alteration. 

The laser beam can be directed along one of several optical paths available in either the upright or inverted microscope configurations. For fluorescence applications, it is often convenient to make use of the standard epi-illumination... 

In one application, an epi-illuminated optical trap was used in conjunction with an inverted fluorescence microscope so that individual cells could be identified with FITC-conjugated antibodies and subsequently trapped for further micromanipulation (24). In the first part of the experiment, as a demonstration of a sorting technique, the trapped cells were transferred into a capillary tube, that was located in the Petri dish along with the cells. The optical trap was used to lift... 

Fig. 7. Schematic diagram of forces exerted on a cell when using an inverted microscope with (A) epi-illumination (i.e., laser focused through the objective) or (B) transillumination (i.e., laser focused through the condenser). is the axial force, and Fl is the lateral trapping force. Curved arrows represent the laser beam waist and point in the direction of light propagation.

In the case of aqueous thin films between oil droplets (Figure 5), the interferometer beam is brought into the microscope through the epi-illumination attachment whereby the objective lens is used to both observe the film and focus the interferometer beam. The contrast of the observed image is much improved in stray light is minimized by positioning a pinhole at the image plan of the epi-illumination device. The thickness calculations remained the same as for the a/w films as the refractive index of the aqueous thin film was the same in both cases. 

Since its development by Delhaye and Dhamelincourt in 1975 [1] the epi-illumination Raman microprobe has become one of the most important input systems in Raman spectroscopy and is the instrument around which most Raman imaging systems are constructed. Epi-illumination instruments are almost always constructed around research-grade commercially available fluorescence microscope frames, with input optics modified to accept an exciting laser and with output optics modified to direct backscattered Raman signal to a spectrograph. 

Total Bacteria Count The TBC is determine by directly counting the actual number of microorganisms collected on a filter after it is used to filter a sample of the water in question.5 The sample is stained with acridine orange and viewed with an epi-illuminated fluorescent microscope. This technique is more accurate and quicker than the culture technique, but is not as practical for field work. 

For single-capillary systems, a conventional PMT is used for detection. Light is routed to that PMT either with fiber optics, a collecting mirror, epi-illumination microscopy, or a microscope objective. For multiplecapillary systems, the system must be scanned [6] or the light imaged onto a CCD camera. 

Figure 5. Experimental setup of a confocal microscope for optical single-molecule detection by epi-illumination.

The cell design shown in Fig. 2c has been used for micro-SERS spectroscopy of chromosomes and related material This microcell is for use in a Raman microspectrometer using epi-illumination. By choice of the objective used in the microscope, the focus of the laser beam is about 6 pm in diameter for a typical chromosome micro-SERS-spectrum. The working electrode, with a diameter of 0.5 mm, is fitted into a perspex rod and can be screwed into a frame for positioning the electrode surface to the objective of the microscope. This small microcell needs only 0.08 ml of sample. 

Fig. 1. TIRF microscope. (A) A view of the TIRF system. (B)View looking down on the back of the microscope (environmental chamber at bottom, back of the system at the top). Arrow b indicates the location of the Selection Prism containing the 80%/ 20% beamsplitter. Pulling the knob at the top to its full upwards position sets the beamsplitter to 100%/ 0% (all Epi-illumination input to the microscope). Pushing it downward sets the beamsplitter to 80% laser and 20% epifluo-rescence illumination. (B ) Location of the Field Diaphragm and the Field Stop on the epifluorescence arm of the split box. Two arrows AS and FS indicate Aperture Stop and Field Stop, respectively. Adjustment of these is vital for good IRM imaging. (B ) The laser input from which the screw white arroW) for TIRF angle adjustment projects. This pair of screws laterally translocates the laser path off center in the objective so that the angle of reflection is altered.

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