Upright microscope

Figure 7.11. T1RF adapted to an upright microscope, shown here with a special sample chamber for long-term viewing of cells in a plastic tissue culture dish. The prism is a truncated equilateral triangle. The region of the sample chamber is shown enlarged relative to the rest of the microscope for pictorial clarity. The sample chamber is provided with a slow flow of humidified 5% C02/air, and 37 °C air is blown over the whole region.

Fig. 2. Diagrammatic summary of the method of mounting and viewing cells in culture dishes on an upright microscope.

The laser light travels through the epifluorescence or side port of the microscope. A dichroic mirror reflects the laser light and passes the green fluorescence to either of the detectors. Detectors are positioned on the bottom port of the inverted microscope or the top port of the upright microscope. The choice of detector is discussed in more detail below. Broadband and band-pass filters placed in the detection path prevent residual IR from reaching either of the detectors. 

The goal of this chapter is to understand the filter sets used for fluorescent microscope so that the best image can be collected. The upright microscope is the most common choice for examining fixed tissue. The bright field microscope used to examine labeling with HRP and its developed chromogens are familiar to most scientists and will not be discussed. 

Upright microscope Specimen placed below the objective lens Tissue slice (histology work) High resolution but short working distance... 

Fig. 9 Schematic diagram of a prism-type TIR setup based on an upright microscope DEP, depolarizer ND, neutral density filter M, mirror L, focusing lens PR, prism PD, petri dish MO, microscope objective BF, barrier filter II, imager intensifier CCD, charge-coupled device...

Place the females in a drop of halocarbon oil or mineral oil on a slide (for imaging on an upright microscope) or on a 22 X 40-mm coverslip (for imaging on an inverted microscope). Pull out the ovaries by grasping the tip of the abdomen with the forceps. 

For analysis on an upright microscope, place small coverslips (18 mm ) on the slide at the sides of the oil to act as supports (secure with double-stick tape) and cover with a 20 X 40-mm coverslip. For analysis on an inverted microscope, place the coverslip on the microscope stand and proceed with imaging. Ovaries in this condition appear healthy for at least 1 hour, allowing time-lapse analysis. 

DESIGN OE MICROMANIPULATION SUPPORT SLIDES, 347 Upright Microscopes, 347 Inverted Microscopes, 348... 

DESIGN OF MICROMANIPULATION SUPPORT SLIDES Upright Microscopes... 

Figure 18.1. Micromanipulation slide designed for upright microscopes. (A) A perspective drawing of the metal microinjection support slide (for a mechanical drawing, see Kiehart 1982). (S) The assembled injection chamber showing top and bottom coverslips in place, the microscope stage, and part of a micropipette. The arrow points to the position of the embryos near the front edge of the bottom side of the top coverslip.

For low-resolution work, we have used a low-NA, dry objective mounted on an upright microscope and have injected embryos mounted on a coverslip resting directly on a glass slide. This strategy is useful if injected embryos are subsequently observed on another microscope (e.g., an inverted, scanning confocal microscope). 

If subsequent manipulations are to be performed on an upright microscope, attach a second coverslip to the micromanipulation slide with a thin layer of vacuum grease. A second coverslip may not be necessary if an inverted microscope is used (see p. 348). 

Cover the embryos with 700 halocarbon oil. For upright microscopes, a coverslip must be used. Generally limit the time embryos are covered by the coverslip to approximately 20-30 minutes. Support the coverslip, if necessary, with a small amount of plasticene at each corner. The coverslip should be lowered as close as possible to the embryo, by pushing down gently on the plasticene at each corner. Do not compress the embryo itself. 

Upright microscope, e.g., Eclipse (Nikon, Tokyo, Japan) equipped with a lOx objective (Plan Fluor, Nikon), and a 60x objective (1.00 NA), water immersion (Plan Fluor, Nikon). 

Brain slices were transferred and anchored to a recording chamber with a nylon grid. A peristaltic pump continually perfused the slices with ACSF (bubbled with carbogen) at a rate of 1-2 mL/min. The recording temperature was controlled at either 26 or 35 °C with an inline heating system. Slices were visualized with an upright microscope equipped with DIG optics and were displayed on a monitor using a CCD camera. 

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