Microscope in the Spotlight

CONTENTS


Selective Plane Illumination Microscopy (SPIM) or Light-sheet (LISH) microscopy

Dr Louise Cole (Bosch Institute, Advanced Microscopy Facility, The University of Sydney) recently attended a light-sheet workshop in Melbourne (organised by Monash Micro Imaging in Feb 2016), together with like-minded microscopists with a keen interest in exploring new techniques. This workshop followed the inaugural 1st Light-Sheet Meeting in Sydney that was held in November 2015 (Meeting Flyer).

Attendees at the 2015 Light-Sheet Meeting

Attendees at 2015 Light-Sheet Meeting at the School of Medical Science, The University of Sydney. Photo: Clive Jeffrey.

Light-sheet (LISH) microscopy or selective plane illumination microscopy (SPIM) is not a new technique; first invented in the 1900s by Siedentopf & Zsigmondy (1903). However, it is only recently that SPIM has become the rising star in the world of imaging technology (Reynaud et al. 2008, Santi 2011 for a review). The recent 2016 MMI/LISH workshop included seminars covering various topics; how to build an open-SPIM, LISH applications and clearing methods as well as hands-on demonstrations of two LISH instruments: the diSPIM (ASI) and an Open-SPIM (open-source hardware platform).

Alignment of the Open-SPIM at the MMI LISH meeting, Feb 2016

Alignment of the Open-SPIM at the MMI LISH meeting, Feb 2016. Photo: Paul Rigby.

SPIM technology delivers fast acquisition with minimal photobleaching and phototoxicity. These attributes are required for imaging larger cleared samples and augment other fluorescence imaging techniques such as spinning-disk, confocal and multi-photon microscopy.

The Bosch Institute Advanced Microscopy Facility (AMF) purchased a commercial light-sheet microscope, the Ultramicroscope II (LaVision BioTec) in 2015. The Ultramicroscope II is suitable for imaging a range of cleared samples (less than 1 cm cubed).


Whole Mouse brain cleared using the PACT-method.

Whole Mouse brain cleared using the PACT-method.

Dopaminergic Neurons

Dopaminergic neurons, in a mouse midbrain (2mm slice) made optically transparent using the PACT technique, were labelled with anti-tyrosine hydroxylase antibody followed by an Alexa-594 secondary antibody (5 days each) and imaged using the LaVision BioTec Ultramicroscope II (see image of instrument below). This maximum intensity projection (using a temporal-colour code LUT, spectrum) reveals neurons throughout the 268-μm Z-slice. Photos: Dr Louise Cole.


The LaVision Ultramicroscope II

The LaVision Ultramicroscope II at the Bosch AMF.

Access to the Ultramicroscope II here at the AMF, is FREE and open to all until the end of October 2016. For training dates, please see the following link (AMF training workshops brochure).


For further information, please contact Dr Louise Cole directly by email: .

Dr Louise Cole is grateful to Anatomy & Histology at the University of Sydney for providing support for attendance at the 2016 MMI/2016 LISH meeting.

Dr Louise Cole
Senior Research Fellow
Bosch Institute Advanced Microscopy Facility
School of Medical Sciences,
The University of Sydney.


ZEISS AxioScan.Z1

AxioScan.Z1

The ZEISS AxioScan.Z1 slide scanning microscope in the Bosch Institute Advanced Microscopy Facility (AMF) has rapidly become a workhorse instrument. With over 40 users already trained on this microscope and 4300 hours of usage recorded since December 2014, researchers are now requesting more time on this microscope.

The ZEISS AxioScan slide scanner can digitize whole slide preparations. Up to 100 slides can be loaded and scanned overnight using the transmitted light or fluorescence mode. Researchers are quickly realising the benefits of imaging and analysing the entire sample on the slide but are now struggling to handle the large data files and are forced to revisit their data management strategies.


Alzheimer Brain

Claude Dennis: post-mortem human brain stained with immunohistochemical marker.

Examples of slide preparations include formalin-fixed paraffin-embedded samples of post-mortem human brain stained with immunohistochemical markers. Claude Dennis (PhD student, Pharmacology) uses the AxioScan to measure the percentage staining, using markers of oxidative stress, in different regions of these samples of Alzheimer-diseased brains compared to controls. He finds the AxioScan very easy to use and that less time spent on the microscope gives him more flexibility and time for image analyses.


Bianca McCarthy (PhD student, Physiology) uses the AxioScan to collect images of whole skin sections that have been irradiated with ultraviolet light (UV). Some of the skin samples are first treated with various topical formulations prior to irradiation. Others are treated with vitamin D metabolites after irradiation. She is interested in whether any of these conditions protect against early markers for skin cancer and photo-ageing. These markers are typically DNA damage and DNA repair enzymes that have been identified using immunohistochemistry.

Irradiated skin section.

Bianca McCarthy: skin section irradiated with ultraviolet light.

“Due to the varying treatments, there can be up to 95 slides that need to be imaged at any one time. Prior to using the AxioScan, all imaging was done manually, which took months. However, now using the AxioScan, the slides can be imaged overnight. Clearly, there are time saving benefits using the AxioScan, which ultimately allows us to find the answer to our question a lot quicker.” Bianca McCarthy


Nasal mucosa biopsy from cat.

Joanna Whitney: nasal mucosa biopsy from cat.

Joanna Whitney (Lecturer in Small Animal Medicine, PhD student, University Veterinary Teaching Hospital) is investigating the immune-pathogenesis of feline upper respiratory tract aspergillosis. She is currently characterising the immune cell populations within the nasal mucosal of affected cats. In order to achieve this, nasal mucosa biopsy samples from affected and unaffected cats are immune-histochemically stained for different inflammatory cell markers and immunoglobulin expression. Due to the small and irregular size of the samples, scanning the slides allows Dr Whitney to easily determine tissue areas where cell counts can be performed and compared.


Dr Anthony Ashton (Scientific Director, Division of Perinatal Research, Kolling Institute of Medical Research) uses the AxioScan to investigate neovascularisation; the process of blood vessel expansion. This process takes place rarely in adult organisms outside of wound healing or reproduction, except for diseases such as cancer, arthritis and blindness. Dr Ashton’s group is currently looking at the molecular regulators of neo-vascularisation to provide therapeutic targets that will improve upon the existing first generation targets to decrease side effects of treatment while enhancing patient outcomes and therapeutic milestones.

Anthony Ashton: investigating neovascularisation.

Anthony Ashton: investigating neovascularisation.

Dr Ashton explains that they were frustrated with quantifying only “representative images” of immunohistochemically stained samples. They require this slide-scanning technology in order to quantify staining without selection bias and to image their samples without spending days on the microscope. They believe this system will provide a platform to circumvent these problems.


Other projects planned for this microscope include the scanning of old Pathology teaching and museum archival slides that need to be preserved digitally.

For more information about how to access the ZEISS AxioScan slide-scanning microscope at the Bosch Institute AMF, please contact Dr Louise Cole ().