A multispectral LED array for the reduction of background autofluorescence in brain tissue - PubMed (original) (raw)

A multispectral LED array for the reduction of background autofluorescence in brain tissue

Haison Duong et al. J Neurosci Methods. 2013.

Abstract

The presence of fixative-induced and cellular-derived artifactual autofluorescences (AAFs) presents a challenge in histological analysis involving immunofluorescence. We have established a simple and highly effective method for the reduction of AAFs that are ubiquitous in fixed mammalian brain and other tissues. A compact AAF-quenching photo-irradiation device was constructed using a commercially available light emitting diode (LED) array, cooling unit, and supporting components. The LED panel is comprised of an array of multispectral high intensity LEDs which serve as the illumination source for the photo-irradiation process. Rabbit and cat brain specimens of 5 μm- and 40 μm-thicknesses, respectively, were photo-irradiated for various durations. The AAFs were reduced to near tissue background levels after 24h of treatment for both deparaffinized and paraffinized rabbit brain specimens, and for the free-floating cat brain specimens. Subsequent immunofluorescence staining using primary antibodies against GFAP, NeuN, Iba-1, and MAP-2, and the corresponding Qdot(®) and Alexafluor(®) fluoroconjugates confirmed that the LED photo-irradiation treatment did not compromise the efficiency of the immunofluorescence labeling. The use of the device is not labor intensive, and only requires minimal tissue processing and experimental set-up time, with very low maintenance and operating costs. Finally, multiple specimens, in both slide and well-plate format, can be simultaneously photo-irradiated, thus, allowing for scalable batch processing.

Keywords: AAFs; Antibody; Autofluoresence; Fluorophores; Immunofluorescence; LEDs; Light emitting diodes (LEDs); Photo-irradiation; UV; artifactual autofluorescences; light emitting diodes; ultraviolet.

Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.

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Figures

Fig. 1

Fig. 1

Assembly and operation of the LED array device. (A) The components include: (1) a cooling housing unit, (2) an LED array panel, (3) aluminum supporting stand, (4a) plate holder, and (4b) slide holder. (B) LED array device assembly with components inside as numbered in (A). (C) Illustration of LED array device operation and corresponding temperature output measured at tissue-to-air interface with and without cooling. The height of the specimen holder rack can be adjusted to vary the distance from the LED panel surface. Plot showed slightly lower temperature at the tissue-to-air interface with additional cooling from the housing unit.

Fig. 2

Fig. 2

Robustness of LED array-based photo-irradiation with multiple tissue specimens in 5 μm-thick paraffin-embedded (left column) and deparaffinized (middle column) rabbit cortical sections, and 40 μm-thick cat cortical sections (right column). Fluorescence intensity of selected AAF spots of interest (SOIs, n = 6) are circled and numbered. Non-AAF tissue background regions are labeled “BKG.” The BKG region on each image is representative of five other regions selected for measurements (not shown for simplicity). Plot showed the mean percent-reduction of SOIs with respect to BKG at each treatment duration (see Eq. (1)). Error bars represent one standard deviation. *Statistical significance (p < 0.05) comparing treatment time to the initial time point of t = 0 h. §Statistical significance (p < 0.05) comparing between treatment durations. Statistical significance was determined by paired Student's _t_-test analysis (n = 6).

Fig. 3

Fig. 3

Reagent buffer control test. Deparaffinized 5 μm-thick slide-mounted and 40 μm-thick free-floating in well-plate specimens were immersed in citrate buffer for 24 h. No difference in AAF reduction was observed demonstrating that the citrate buffer reagent did not contribute to AAF's reduction.

Fig. 4

Fig. 4

Reduction of AAFs in a 40 μm-thick cat brain specimen pre- and post-treatment by the LED array. (A) Laser-scanned image of a pre-treatment specimen showing AAF presence in individual spectral channels, and (B) throughout the entire thickness of the specimen (_Z_-axis view). (C) The elimination of AAFs across the entire spectrum following treatment on the same specimen, and (D) shows elimination of AAFs throughout _Z_-axis. The specimen was photo-irradiated for 24 h at a distance of 7 mm from the LED panel surface. A staining integrity of the post-treatment specimen was demonstrated in Fig. 5.

Fig. 5

Fig. 5

Quality of triple-label immunostaining after 24-h-treatment on the same 40 μm-thick cat brain specimen as shown in Fig. 4D, using primary antibodies against Iba-1 (microglia), GFAP (astrocytes), NeuN (neural cell bodies) and corresponding AlexaFluor® fluorophores: Iba-1 (488 nm), GFAP (633 nm), and NeuN (546 nm). Image was obtained on LSM 510 laser scanning system. False colors were applied for each fluorophore. Iba-1, red; GFAP, green; NeuN, magenta. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 6

Fig. 6

Immunostaining quality comparison between untreated and treated 5 μm-thick rabbit brain specimens. Specimens were photo-irradiated for 4 h at a distance of 1 mm away from the LED panel surface. The large hole represents an electrode shaft area with AAFs. (A) GFAP staining (Qdot® 655 nm, red) on untreated specimens with AFFs (white arrows). (B) GFAP staining (Qdot® 655 nm, red) on treated section showing no AAFs present. (C) Triple staining for GFAP (Qdot® 585 nm, yellow), NeuN (Qdot® 525 nm, green), and MAP-2 (Qdot® 655 nm, red) following treatment. Note the absence of AAFs in the treated groups (B and C). Samples were imaged on a widefield fluorescence scope (Olympus Inc.) using a multiband excitation (495 nm) and emission filter (520–700 nm) for the Qdot® fluorophores used. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 7

Fig. 7

Reduction of AAFs in multiple spectral channels in confocal images following 24 h treatment on two vertically adjacent 40 μm-thick cat brain specimens. (A) Image showed AAFs present (circles) in multiple channels prior to treatment, and (B) subsequent quadruple-labeled immunostaining using primary antibodies and AlexaFluor®fluorophores for Iba-1 (cyan), NeuN (red), GFAP (green), and MAP-2 (dark blue). False colors were applied and some AAFs were detected in the same channels as the labeling fluorophores. (C) Image showed elimination of AAFs in spectral channels following 24-h photo-irradiation on a vertically adjacent tissue section to that of (A), and (D) no interfering AAFs were detected in subsequent immunostaining. Images were obtained on an LSM 510 laser scanning system. The specimens were photo-irradiated for 24 h at a distance of 7 mm from the LED panel surface. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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