Confocal Microscopy Assessment Research Articles

Correlative electron and confocal microscopy assessment of synapse localization in the central nervous system of an insect

Excellent methods exist to analyze sub-neuronal structures, such as synapses, at nanometer resolution with electron microscopy. However, due to methodological constraints, electron microscopy is feasible only for small volumes of fixed tissue. By contrast, confocal or two-photon laser scanning microscopy is well suited to obtain neuronal structures from large volumes of living or fixed tissue at sub-micrometer resolution. Therefore, a gap exists when analyzing synaptic organization of neuropils, or the distribution of synapses throughout the dendritic trees of individual neurons, and it would be advantageous to use confocal microscopy to investigate the synaptic organization of central neuropils. This study uses correlative electron and confocal microscopy from the same tissue sections to test whether synapsin I-immunopositive puncta can be analyzed at the light microscopy level to estimate the distributions of synaptic sites within central motor neuropils and along reconstructed dendritic surfaces in an insect ventral nerve cord. It demonstrates that every type 1 synaptic terminal can be detected as a distinct punctum by synapsin I-immunolabeling and confocal microscopy. Furthermore, it provides data indicating that co-localization analysis from confocal image stacks as recently published provides a good estimate for quantifying the distribution patterns of input synapses through dendritic trees.

Quality assessment of confocal microscopy slide‐based systems: Instability

All slide-based fluorescence cytometry detections systems basically include an excitation light source, intermediate optics, and a detection device (CCD or PMT). Occasionally, this equipment becomes unstable, generating unreliable and inferior data. A number of tests have been devised to evaluate equipment performance and instability. The following four instability tests are described: galvanometer scanning, stage drift, correct wavelength spectral detection, and long-term laser power. Quality assurance tests revealed that a confocal microscope can become unstable in the following parameters, yielding inaccurate data: laser power, PMTs functionality, spectrophotometer accuracy, galvanometer scanning and laser stability, and stage drift. Long-term laser power stability has been observed to vary greatly. Confocal systems can become unstable in the following parameters: long-term laser power, galvanometer scanning, spectrophotometer accuracy, and stage stability. Instability in any of these parameters will affect image quality. Laser power fluctuations result from either a defective Acousto-optic tunable filter or improper heat dissipation. Spectrophotometer instability will generate unreliable spectra data, extra light reflections, and poor image quality. Galvanometer scanning instability yields poor image quality while microscope stage drift results in a sample going out of the plane of focus. With minor modifications, these tests may be applicable to other slide-based systems.

5% Imiquimod Cream and Reflectance-Mode Confocal Microscopy as Adjunct Modalities to Mohs Micrographic Surgery for Treatment of Basal Cell Carcinoma

Imiquimod is an immune response modifier that up-regulates cytokines and has been shown in clinical studies to reduce or clear basal cell carcinoma tumors when applied topically. The objectives were to evaluate the efficacy of 5% imiquimod cream in treating basal cell carcinoma preceding excision by Mohs micrographic surgery and to determine if reflectance-mode confocal microscopy is useful to establish the need for surgical intervention after imiquimod treatment. Subjects applied study cream to one biopsy-confirmed basal cell carcinoma tumor 5 x/week for 2, 4, or 6 weeks in this vehicle-controlled, double-blind study. Confocal microscopy was used for the 6-week treatment group to examine the target tumor area at each interval visit and immediately before Mohs micrographic surgery. After the Mohs micrographic surgery excision, the tissue was evaluated histologically, and the excision area was measured. Confocal microscopy readings were correlated to the histologic diagnosis. Tumors cleared or the target tumor area was reduced in subjects in the 4- and 6-week dosing regimens. Confocal microscopy assessments correlated well with the histologic diagnosis. Imiquimod improved excision results relative to vehicle when used for treating basal cell carcinoma before Mohs micrographic surgery. Confocal microscopy assessments correlated well with tumor response to therapy, suggesting that confocal microscopy may help determine the need for surgery.

5% Imiquimod Cream and Reflectance-Mode Confocal Microscopy as Adjunct Modalities to Mohs Micrographic Surgery for Treatment of Basal Cell Carcinoma

BACKGROUND Imiquimod is an immune response modifier that up-regulates cytokines and has been shown in clinical studies to reduce or clear basal cell carcinoma tumors when applied topically. OBJECTIVE The objectives were to evaluate the efficacy of 5% imiquimod cream in treating basal cell carcinoma preceding excision by Mohs micrographic surgery and to determine if reflectance-mode confocal microscopy is useful to establish the need for surgical intervention after imiquimod treatment. METHODS Subjects applied study cream to one biopsy-confirmed basal cell carcinoma tumor 5 ×/week for 2, 4, or 6 weeks in this vehicle-controlled, double-blind study. Confocal microscopy was used for the 6-week treatment group to examine the target tumor area at each interval visit and immediately before Mohs micrographic surgery. After the Mohs micrographic surgery excision, the tissue was evaluated histologically, and the excision area was measured. Confocal microscopy readings were correlated to the histologic diagnosis. RESULTS Tumors cleared or the target tumor area was reduced in subjects in the 4- and 6-week dosing regimens. Confocal microscopy assessments correlated well with the histologic diagnosis. CONCLUSION Imiquimod improved excision results relative to vehicle when used for treating basal cell carcinoma before Mohs micrographic surgery. Confocal microscopy assessments correlated well with tumor response to therapy, suggesting that confocal microscopy may help determine the need for surgery.