Confocal Microscopes

In preparation for cryo-confocal microscopy samples are rapidly frozen to low temperatures (typically below -150°C) using liquid nitrogen or other cryogens. Cryogenic freezing helps protect structural integrity of the samples and prevents ice crystal formation. The cryo environment stabilizes samples preserving native structures, reduces photobleaching and photodamage, and enables imaging of hydrated samples without dehydration or fixation. Cryo-confocal microscopy can be combined with cryo-electron microscopy (Cryo-EM) for correlative studies (CLEM). Cryo CLEM enables detailed visualization and analysis of samples in a near-native state, providing valuable insights into the organization and ultrastructure of proteins and macromolecules.

STED super-resolution microscopy allows imaging at resolutions well below the diffraction limit based purely on physical principles. The key concept of STED relies on the ability to control the states of a fluorophore, i.e., emitting vs. dark states. The emission of fluorophores within a diffraction-limited spot is confined to a sub-diffraction region by overlaying a donut-shaped laser beam of appropriate wavelength (STED beam) onto the excitation beam of a confocal microscope. This approach forces the fluorophores under the effect of the STED beam to return to the ground state before spontaneously emitting a photon. The effective focal volume can be reduced up to a few tens of nm. For more information, refer to: Nanoscopy Meets Lifetime - Introducing the new TauSTED

Fluorescence lifetime is a measure of how long a fluorophore remains in its excited state before returning to the ground state by emitting a photon. The characteristic time of this emission measured on a population of fluorophores is called  fluorescence lifetime, which is in the range of picoseconds to nanoseconds. Fluorescence lifetime is a characteristic parameter of a given fluorophore that may change with its local environment or conformational state, while remains independent of the fluorophore concentration. Local environmental factors are ion concentration, pH, lipophilicity, or the presence of other molecules close to the fluorophore. This fact makes FLIM ideal for functional imaging which enables investigation of molecular function and interactions. Additionally, FLIM can be useful for distinguishing fluorophores with overlapping emission spectra or eliminating unwanted background fluorescence. For more information on FLIM, refer to: Fluorescence Lifetime Imaging

When imaging deep inside thick specimens and samples, performing confocal fluorescence microscopy with one-photon excitation can be challenging due to the scattering of visible light. The maximum imaging depth achievable for one-photon excitation is around 100 µm. In contrast, multiphoton microscopy takes advantage of multiphoton excitation with infrared light which has reduced scattering due to the longer wavelengths. This fact makes multiphoton microscopy ideal for deep tissue imaging of thick specimens and samples. Multiphoton microscopy has been used for such things as visualizing the complex architecture of the whole brain as well as the study of tumor development, metastasis, and immune response in organisms. For more details, refer to the tutorial: Principles of Multiphoton Microscopy for Deep Tissue Imaging

Light-sheet microscopy, also referred to as selective-plane illumination microscopy, is a gentle way of imaging sensitive samples or fast biological processes in live specimens. The optical scanning is only done in a single plane and detected from the perpendicular direction. The excitation is done with a thin light-sheet that illuminates the focal plane and spares the sample from out-of-focus excitation. DLS microscopy uses a digital camera and is advantageous for fast imaging of live specimens. For more details, refer to the article: Confocal and Digital Light Sheet Imaging

A microscopy method which takes advantage of the intrinsic vibrational contrast of molecules within biological specimens. It is normally done in 2 ways: either with coherent anti-Stokes Raman scattering (CARS) or stimulated Raman scattering (SRS). The big advantage of CRS is that no labeling of the specimen is required, because the image contrast arises from the spectroscopic properties of various molecular species present in the specimen. As there is no labeling, CRS can allow specimens to be imaged in a nearly pristine state. For more information, refer to: Coherent Raman Scattering (CRS) Microscopy

It is an optimal light source for confocal microscopes. The white light laser (WLL) consists of a high-energy pulsed IR-fiber laser that is fed through a photonic crystal fiber to generate a spectral continuum. Small bandlets are selected from that continuum by means of an acousto-optical beam splitter (AOBS). The WLL provides excitation tunable from blue to red throughout the spectrum. For more details, refer to the article: STELLARIS White Light Laser

For fluorescence microscopy, it is desirable to filter and separate specific color bands for the excitation and emission of fluorophores. In the past, filter and beam splitting was usually conducted with planar optical elements like filters and mirrors. They have the limitation of fixed specification and slow exchange. An entirely different approach is the employment of acousto-optical elements for both excitation control, done with an AOTF, and excitation emission separation, done with an AOBS. These acousto-optical devices allow flexible tunability and high-speed switching. For more details, refer to the article: Acousto Optics in True Confocal Spectral Microscope Systems