Imaris for Beginners

Imaris is a powerful 3D/4D analysing program that registered users of MMI has access to. To boost the usability of this program I have organised a 2-day beginner's course that is being run by the creators of Imaris, Bitplane. The course will be held on the 22/23rd of November and will cost $400 for the two days, which include morning/afternoon tea and lunch. This course will provide you with the confidence to use Imaris along with all the tools you will need to collect meaningful and statistically significant data from you own research.

Examples of what Imaris is currently being used for by MMI uses include:
> measuring volume and number of vesicles within a cell
> measuring spindles during mitosis (length, number, volume etc)
> measuring co-localisation of proteins within cells
> and many more....

If you would like to attend the course, please contact me via email (alex.fulcher@monash.edu).

Preparation Of Mowiol Mounting Medium

Preparation of Mowiol Coverslip Mounting Solution for Fluorescence Microscopy

This protocol describes the preparation of a coverslip mounting solution called Mowiol. Mowiol (Mowiol 4.88, Calbiochem catalog number 475904) is a solution of polyvinyl alcohol. This solution normally hardens overnight after slide preparation, and does not require the coverslips to be sealed with nail polish. Put 6 g glycerol in a 50 ml plastic centrifuge tube and add a small stir bar.

1. Add 2.4 g Mowiol (Calbiochem); stir to mix.

2. While stirring, add 6 ml distilled water and leave 2 hours at room temperature.

3. Add 12 ml 0.2M Tris (pH 8.5).

4. Add a small amount of NaN₃ (Sodium Azide) such that the final concentration is 0.02% (optional).

5. Incubate the tube in hot water (50 – 60ºC) for 10 minutes to dissolve the Mowiol. This can be repeated over several hours if necessary.

6. Centrifuge at 5000 g for 15 minutes to remove any undissolved solids. Store 1ml aliquots in eppendorf tubes at -20ºC.

7. Warm tubes to room temperature for use. Opened tubes can be stored at 4ºC for approximately 1 month. Discard if any crystalline material is seen in the tube or on the slides.

8. Leave cover slipped slides in the dark overnight to harden before oil immersion lenses are used or put at 37oC for about 10 min.

Note: Do not use so much mounting solution that the coverslips are floating. Normally, ~5 µl for a 10mm round coverslip, 15 – 20 µl is sufficient for a 22 x 22 mm coverslip. 22 x 50 mm coverslips require about 40 – 50 µl.

Benchtop Preparation for TEM

Transmission electron microscopy (TEM) can be a fantastic way to obtain a wealth of information about biological structures and processes. Unfortunately the conditions inside a TEM are not particularly hospitable for biological tissues. To successfully pass electrons through TEM specimens, they must be thinner than a wavelength of visible light, resistant to hard vacuum and completely dehydrated. (In other words: dead!)

Preparing biological specimens for TEM therefore requires preservation, dehydration and embedding of the sample for sectioning on an ultramicrotome. Specimen preparation is arguably the most important factor in acquiring high quality images, so here is a generic (“benchtop”) protocol that we use as a guide when preparing samples for ultra-thin sectioning. See below the protocol for important notes... but first...

DISCLAIMER: This protocol is meant strictly as a guide for persons with a high level of technical competency. I do not provide a warranty of any kind for the quality of specimens prepared using it, and I cannot be held responsible in any way. Do not use this guide if you are unqualified or unsure. Read the MSDS for all chemicals and reagents before use, and use the correct personal protective equipment. Fear osmium, because it can kill you.

Generic Benchtop Specimen Preparation:

Step	Reagent	Time
Primary Fixation	4% PFA in Buffer	1 hour
Secondary Fixation	2.5% GA in Buffer	1 hour
3-5x Buffer Wash	0.1M Buffer	10 min
Tertiary Fixation	1% OsO4 (aq) in Buffer	1 hour
1-2x Buffer Wash	0.1M Buffer	10 min
3x Water Wash	MilliQ Water	10 min
En Bloc Staining	2% Uranyl Acetate	1 hour
3x Water Wash	MilliQ Water	10 min
Dehydration	50% Ethanol	10 min
Dehydration	70% Ethanol	10 min
Dehydration	90% Ethanol	10 min
Dehydration	100% Ethanol	10 min
Dehydration	100% Ethanol	10 min
Dehydration	100% Transition Solvent	15 min
Dehydration	100% Transition Solvent	15 min
Infiltration	Solvent:Resin (3:1) [25%]	1 hour
Infiltration	Solvent:Resin (1:1) [50%]	1 hour
Infiltration	Solvent:Resin (1:3) [75%]	1 hour
Infiltration	Resin 100%	1.5 hour
Embedding	Resin 100% @ 60-70°C	24 hour

Common acronyms:

PFA = Paraformaldehyde

GA = Glutaraldehyde

OsO₄ = Osmium Tetroxide

Reagent Note:

The buffers, transition solvents and resins are often tailored to specific projects. A good starting point would be 0.1M Phosphate Buffer, Acetone and Epon-Araldite (aka. Procure-Araldite).

Debbie Symons & Jasmine Targett: Making Sense

Debbie Symons & Jasmine Targett: Making Sense

Works include a collaboration between Jasmine Targett, Judy Callaghan (MMI) and Alina Donea (Monash Centre for Astrophysics)

 

Exhibition:  6-8pm, 9th Sept -15th Oct 2011,  

Location: Gallery 2, Craft Victoria, 31 Flinders LANE, Melbourne, VIC 3000

Opening speaker: Kit Wise, Associate Dean Teaching & Learning, Department of Fine Arts, Faculty of Art & Design, Monash University.

Live Cell Imaging Course 2011

Monash Micro Imaging will be holding its annual live cell imaging course LCI2011 from 27th November until the 2nd December

First Post

Need an Imaris tutorial?
How about an obscure TEM specimen preparation protocol?

Then welcome to the Monash Micro Imaging (MMI) blog.

This blog will provide news, tutorials and information straight from the staff of MMI.