Analyzing Biological Parameters Using Laser Scanning Microscopy

Biomedical sciences are one of the key future-oriented research fields and provide new insights into the complex mechanisms of live systems at the tissue, cellular and molecular levels using robust technologies.

Multiphoton microscopy is believed to be the ideal technique in the minimal and non-invasive fluorescent microscopy field. The laser scanning microscopes LSM 710 NLO and LSM 780 NLO from Carl Zeiss enable researchers to create images of very deep lying tissues with sub-cellular resolution.

LSM 710 NLO and LSM 780 NLO

The LSM 710 NLO and LSM 780 NLO in combination with the Axio Examiner from Carl Zeiss provide the optimal multiphoton system for electrophysiological research and intravital imaging.

Together with the Axio Observer, an inverted research microscope platform from Carl Zeiss, the systems provide a unique multifunctional instrument for the imaging of standard specimens and cells in culture (Figures 1 and 2).

Analyzing Biological Parameters Using Laser Scanning Microscopy

Analyzing Biological Parameters Using Laser Scanning Microscopy

3D Morphology

Functional interconnections particularly in brain tissue can be well understood by knowing the morphology. Sub-cellular structures such as spines and thin axonal processes require imaging with high signal to noise ratio and a large volume or area because of the extensive cell processes (Figure 3).

Illustration of the XZ level of a homogeneous colored sample after laser excitation in the visible range (1), using a multiphoton laser (2), and alternative detection using NDD (3).

Figure 1. Illustration of the XZ level of a homogeneous colored sample after laser excitation in the visible range (1), using a multiphoton laser (2), and alternative detection using NDD (3).

The LSM 710 NLO or LSM 780 NLO meets these technical requirements by extensively blocking the excitation light and through the efficient light guidance of the signals towards the detectors. The systems, featuring GaAsPtype detectors with twofold higher QE and lower dark noise than traditional PMT detectors, convert fluorescent signals into 3D images, which will provide new insights (Figure 4).

A comparison of the intensity distribution along the Z-axis shows the noticeably better excitation in deeper layers of the specimen using the multiphoton laser. It also shows the more efficient signal acquisition using non-descanned detectors.

Figure 2. A comparison of the intensity distribution along the Z-axis shows the noticeably better excitation in deeper layers of the specimen using the multiphoton laser. It also shows the more efficient signal acquisition using non-descanned detectors.

Intravital Imaging

Minimal impact research needs to be performed on live specimens to know interactions and functional connections of cells inside organisms. The point excitation using a pulsed infrared (IR) laser is minimally invasive with phototoxicity at a low level, thus creating the suitable environment for the analysis of live specimens.

The IR excitation light enters deeper into tissues thanks to low scattering, enabling the visualization of even sub-cellular structures in great depth. The concurrent use of various dyes and channels enables the analysis of up to five signal types and consequently the interactions between various structures.

Magnified section of the projection neurons’ dendritic branches.

Figure 3. Magnified section of the projection neurons’ dendritic branches.

The unique systems can also perform difficult methods like two-photon uncaging together with calcium imaging (Figure 5). This locally defined manipulation helps in the analysis of physiological processes and interactions (Figure 6).

Neuromuscular junctions in sternomastoid muscle of an adult transgenic mouse that expresses YFP in all motor neurons. Image was acquired in a living animal using the Zeiss W-Plan Apochromat 20×/1.0 NA dipping objective and two photon excitation (880 nm).

Figure 4. Neuromuscular junctions in sternomastoid muscle of an adult transgenic mouse that expresses YFP in all motor neurons. Image was acquired in a living animal using the Zeiss W-Plan Apochromat 20×/1.0 NA dipping objective and two photon excitation (880 nm). Stephen Turney, MCB, Harvard University, USA

3D in Temporal Resolution

The point excitation of multiphoton systems considerably reduces phototoxicity because the impairing effect of the light is only in the focus. In embryology, this technique enables researchers to extensively analyze developmental processes, including cell organization and cell distribution. The LSM 710 NLO and LSM 780 NLO enable the optimal investigation of the behavior of immuno-active cells either in an artificial 3D collagen matrix or in vivo for as long a duration as possible (Figure 7).

3D reconstruction of a zebrafish embryo expressing a genetically encoded Ca2+ indicator, Cameleon. Early developmental stages of the embryo were observed for 13 hours at 25° C. Excitation at 850 nm, timestamp post fertilization.

Figure 5. 3D reconstruction of a zebrafish embryo expressing a genetically encoded Ca2+ indicator, Cameleon. Early developmental stages of the embryo were observed for 13 hours at 25° C. Excitation at 850 nm, timestamp post fertilization.

Second Harmonic Generation (SHG)

SHG is a non-linear photophysical effect utilized in non-linear microscopy for the creation of additional contrast. In this process, a strong incident laser’s two photons are driven through polarizable tissue and converted into a single photon with twofold energy and frequency levels.

SHG does not require dyes and seeing as the image contrast is structurally inherent to the sample (Figure 8). Hence, Second Harmonic Imaging is an ideal technique to explore live tissues and cells. The additional contrast presents key data on the structure and/or changes found in some proteins. The LSM 710 NLO or the LSM 780 NLO on Axio Examiner is suitable for this application thanks to its special optics.

Second Harmonic Imaging of embryonic stem (ES) cell-derived mouse motor neurons in vitro. The motor neurons were established in a long-term co-culture (5 days) with either ES cell-derived or primary glial cells. The image is a composite of SHG (B) and oblique illumination contrast (A) signals acquired simultaneously using low-intensity multiphoton excitation (800 nm).

Figure 6. Second Harmonic Imaging of embryonic stem (ES) cell-derived mouse motor neurons in vitro. The motor neurons were established in a long-term co-culture (5 days) with either ES cell-derived or primary glial cells. The image is a composite of SHG (B) and oblique illumination contrast (A) signals acquired simultaneously using low-intensity multiphoton excitation (800 nm). Specimen provided by Monica Carrasco, MCB, Harvard University, USA

Sensitivity of the LSM 710 NLO and LSM 780 NLO

The LSM 710 NLO and LSM 780 NLO provide true-to-detail and high-contrast images as they have superior sensitivity together with sophisticated techniques to suppress laser light excitation. Improved non-descanned detectors (NDD) together with the GaAsP technology and better light collection efficiency ensure superior imaging results in thick tissue samples and live animals (Picture 9).

GaAsP NDD Detector for Axio Examiner 1. Beam Splitter 2. Gathering Lens 3. Deflection Mirror 4. Focusing Lens 5. GaAsP Detector

Figure 7. GaAsP NDD Detector for Axio Examiner 1. Beam Splitter 2. Gathering Lens 3. Deflection Mirror 4. Focusing Lens 5. GaAsP Detector

This information has been sourced, reviewed and adapted from materials provided by Carl Zeiss Microscopy GmbH.

For more information on this source, please visit Carl Zeiss Microscopy GmbH.

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