4_photonic Microscopy (tools) : Inverted microscope & phase contrast

The lecturer explains the challenges of using traditional microscopes for cell culture, primarily the need for the objective lens to be close to the sample for maximum resolution. This proximity creates issues with cell culture dishes, requiring either large dishes or water immersion objectives to prevent the cells from drying out. Multi-well dishes, except for six-well dishes, often lack sufficient space for objective insertion. Maintaining sterility during objective insertion is also a concern. The solution presented is the inverted microscope, where the objective lens is positioned below the sample, allowing for close proximity without compromising the culture. The condenser is positioned at a distance, enabling easier access and maintaining sterility. The lecturer describes the inverted microscope's design, highlighting the placement of the light source, collector, condenser, sample (on the specimen stage), and objective lens, emphasizing its practicality for cell culture applications.

Microscope Components and Functionality (00:01:55 - 00:02:54)

The lecturer describes the components of a standard upright microscope, including the light source, condenser, specimen stage, objectives on a turret, beam splitter, oculars, and focusing dials. The condenser's adjustability is noted as dependent on its type. The lecturer explains that this type of microscope is commonly used in cell culture. A component on top of the microscope, used to generate contrast for viewing living (transparent) cells, is identified as a phase contrast mechanism. The need for contrast is highlighted due to the transparency of living cells, which makes them difficult to observe without additional techniques.

Phase Contrast Microscopy Explained (00:02:54 - 00:03:30)

The lecturer introduces phase contrast microscopy as a method to generate contrast in living, unstained cells. The explanation begins by contrasting stained cells (where specific wavelengths are absorbed by the dye, creating contrast) with unstained cells. In unstained cells, no wavelengths are selectively absorbed. However, differences in refractive index between water, cytoplasm, and glass cause phase shifts in the light passing through the cell, which the phase contrast mechanism utilizes to generate contrast.

Phase Contrast Microscopy: Principles and Applications (00:03:30 - 00:04:54)

The lecturer explains the principles of phase contrast microscopy. When light passes through a medium with a higher refractive index (like a cell), it slows down and changes phase. A phase contrast device uses this phase change to generate contrast. This involves an annular ring that focuses part of the light through the sample and a phase ring that modifies the light's intensity, creating contrast. The lecturer compares bright field microscopy to phase contrast microscopy, showing that phase contrast reveals significantly more detail, particularly in visualizing cellular structures like filopodia and lamellipodia. However, a drawback of phase contrast is the halo effect, which can obscure details, especially in thicker parts of the sample like cell bodies. The lecturer notes that more modern phase contrast techniques aim to reduce this halo effect. Examples of images from both bright field and phase contrast microscopy are shown to illustrate the differences in detail and the presence of the halo effect in phase contrast images.

Phase Contrast Microscopy in Cell Culture (00:04:54 - 00:05:24)

The lecturer discusses phase contrast microscopy, noting that while it adds to the cost of a device, it's a relatively inexpensive way to generate contrast in unstained samples. Therefore, it's a standard feature in microscopes used for cell culture. The use of vital dyes is also mentioned; while they allow for staining of live cells, they are ultimately cytotoxic, albeit slowly. This slow killing effect makes them unsuitable for repeated observations of cell cultures over several days, as the dye would impact the results.

Project Kickoff Meeting (00:00:00 - 00:00:00)

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