神经科学: 脑切片机、行為分析、疼痛研究

 

VF200/300 &600 Compresstome™ microtome新一代振动切片机--在电生理及组织切片培养上的巨大优势

新一代振动切片机-美国Precisionary公司

faster, simpler, more productive slicing 

在电生理及组织切片培养上的巨大优势

 AHMSIC 2008 (新一代振动切片机).doc

产品性能

电生理实验需要活的软组织切片,实验的质量取决于切片的质量。脑切片的生存能力,尤其是表层神经元的活性是脑片电生理实验成功的关键因素。美国PI研发的艺术级切片机 VF-200/300可为这些研究提供最高质量的新鲜组织切片,满足不同的研究目的.

● 双倍脑片生存能力(专利技术);

●非常柔软的组织和中空器官切片对于传统的振动切片机很难完成,但对于VF系列来说则没有任何困难---年长的动物的脆弱脑组织和最敏感的脑核区域也可以用VF-200切片,极大的延展了实验的空间;

● 光洁的切片表面--波形痕迹和振动痕迹指示了刀具的轨迹,在VF-200切片表面,振动痕迹令人注目的减少了;

● 高质量图象--VF-200的切片图象如同模板,更少的擦痕和变形,更多的信息被观察;

● 减少热冲击--32C近体温时能够达到和4C切片一样的效果,近体温切片能够消除温度波动,减少对组织的热冲击;

●快速切片痊愈能力 -- 近体温条件下的切片痊愈时间小于20分钟

● 均一的切片厚度--独特的固定及箱位装置最小化样品在三维方向的移动,切片厚度的波动<5%,而传统切片的厚度波动在10-20%;  

●快速切片 -- 传统的振动切片机每层切片的时间在10-30秒,VF-200的切片时间2-3;

 

VF-300是一款高级的可低温保存的显微镜用薄片切片机.

   特别为floating section 免疫组化 大量固定组织切片设计的. 因为其先进的性能,成为floating section 研究的首选机型.

产品优势:

  • 操作简单,节省时间和费用: VF-300 操作只有三步, 组织固定, 组织切片 和 floating staining.     无须过夜蔗糖培养, 无须 OCT 混合埋藏, 无须液氮冷冻. 节省时间和费用.
  • 获得更多更好的信息: 冷冻-解冻 步骤能够降格蛋白和它们的抗原性. 对于那些低水平的信号,用低温保持器更容易得到假阴性的结果。
  • 完美的组织图象. VF-300切片没有冰晶结构.细胞结构和组织图象可以被更好的保持.
  • 切片无破裂. 在染色和制作过程中切片不会破裂
    更深层的EM研究成为可能, 在切片上将有更重要的发现.
  • 全自动推进,无须停止监视,无须手轮摇动来关注每一个切片,减少工作压力。
  • 更宽的切割范围. 低温保持器型的切片厚度大约 5 – 40 um. VF-300 能够切片厚度从10 um 开始无上限.
  • 规则的双刃刀片,便于更换.
  • 温度不受限制. 可在0 37o C温度内高质量切片;
  • 快速切片, 一致的切片厚度. 克服了在振动切片的两个主要的不足.
  • 多功能性. VF-300 能够切片新鲜的组织, 用于精密切片培养或脑片记录.

技术指标:

前进速度:

0-20 mm / s, 可调

返回速度:

20 mm / s, 固定

振动频率:

0-20 Hz, 可调

振幅:

2 mm, 固定

Z-轴颤动:

< 2 µm

刀片:

双刃刀

切角:

13 , 固定(标准)

厚度调节:

数字调节,自动

微分尺:

1 µm / .

最大组织直径:

12.5 mm (标准管), 15.5 mm (大管)

最大组织长度:

25 mm

最小切片厚度:

10 µm

切片模式:

单片/连续

浴槽 :

140 x 60 x 30 mm

电源:

DC 13-15 V

功率:

4 W

尺寸 ( L x W x H ):

330 x 240 x 190 mm

重量:

7 kg

 

Compresstome™ microtome

Compresstome™ is a new family of microtomes developed from our company’s patented technique. The unique features of this technique are specimen embedding and tissue compression just before tissue slice is made, allowing for much thinner slices to be cut with minimal effort and better image quality.

Biological specimen, live or fixed, is glued on the plunger of a specimen tube and embedded with low gel point agarose within the tube. The front end of the specimen tube is smaller to form a compression lip towards the agarose-specimen block. When agarose-specimen block is pushed forword by the plunger, it is compressed by the compression lip. The extruding specimen is sectioned by a sharp blade at the front end of the specimen tube. Much better slice quality is achieved when specimen is compressed. The compresstome™ family of microtmes overcome the specimen move/wobble problems and produce high quality slices with excellent viability, consistent slice thickness, flat, and clean surfaces with sharp image.

The compresstome™ family includes four members; the VF-200, VF-300 and VF-600. The compresstome™ VF-200 is designed for electrophysiology experiments. It is a semi-automatic microtome with manual slice thickness adjustment through a micrometer. It is economical and very effective for cutting small quantity of slices. The compresstome™ VF-300 and VF-600 are fully automatic microtomes. They are suitable for preparing large quantity of slices. Within the three, the compresstome™ VF-300 is for live tissue sectioning. The VF-600 is the model for fixed tissue sectioning.

Key features

Very soft tissues and hollow organs are difficult to cut with vibrating slicers. The Compresstome™ VF-200, and VF-300 microtomes can cut these tissues without any difficulties.

Embryonic brain

Embryonic brain is very soft even in icy cold temperatures. Therefore, it is very difficult to cut them into smooth and even slices. With our patented technique, the embryonic brain is stiffened by the compression and is much easier to slice. The VF-family slicers can overcome similar problems in other very soft tissues such as pancreas, skeletal muscles, and intestines.

Warm temperature slicing

Brain tissues are very soft in warm temperature. They are routinely cut in icy cold buffer with vibrating slicers. With the VF-slicers, you can cut brain slices in temperatures close to their physical environment (32° Celsius). Cutting tissues in warm temperature reduces thermal shock to the neurons during slicing, requiring less recovery time and results in healthier slices.

Wet SEM

The VF-200 is the best slicer that has been tested by Quantomix for Wet tissue scanning electron microscopy. Wet SEM requires slices with a very clean and flat surface to keep close contact with the membrane of the specimen chamber. The VF-200 is the only microtome in the market that meets this standard.

Electrophysiology

Electrophysiology requires slicing of live soft tissues. The quality of your experiment depends on the quality of the tissue slice. Your daily productivity also depends on the quality of the tissue slice. The viability of the brain slice, especially the healthy neurons in the top surface layers are crucial to the success of brain slice patch clamp electrophysiology. The Compresstome™ VF-200 and VF-300 produce the highest quality brain slices for these researches.

Our products have tremendous advantages:

Doubling the brain slice viability.

The pre-stress force created by the compression counteracts the mechanical stress of the cutting blade which reduces cell damage during slicing, resulting in healthier slices with more viable cells. There are many good neurons on the top surface that can be chosen for experiment. Greatly increases your productivity.

Older animal and more sensitive brain areas.

Vulnerable tissues of older animals and more sensitive nucleus of the brain can still be sliced with the Compresstome™ VF-200 and VF-300, greatly expanding the range of experiments that you can do.

Smoother and cleaner cutting surface.

The wavy chatter marks or vibration marks indicate the trails of the oscillating blade of the slicer. In the VF-200 and VF-300 microtome, the vibration mark is dramatically reduced from the slice surface.

High quality images.

Images of slices cut with the Compresstome™ VF-200 and VF-300 are sharper ,with fewer scratches and distortion. As seen from the sample images on the slice album page, more details can be observed in these slices.

Reduce thermal shock.

Slicing can be performed in warm temperature (32 degrees C) as well as 4 degrees C. The ability to slice in warm temperature eliminates drastic temperature fluctuation, reducing thermal shock to the tissues.

Quick recovery.

The ability to slice in warm temperature allows recovery in less than 20 minutes.

Consistent slice thickness.

The unique specimen embedding, holding and clamping mechanisms minimizes specimen movement in all three dimensions during slicing. Consistent slice thickness can be achieved. Slice thickness varies less than 5 percent of the slice thickness (250 µm), compared to 10 to 20 percent in classic slicers.

Minimal moving parts and quiet operation.

Every effort is used to minimize mechanical vibration on the Compresstome™ microtome. This allows them to reduce energy precipitating on the slice.

Versatile.

Need to do some immunohistochemistry work as well? The VF-200 and VF-300 are also great at slicing fixed tissues, saving your money and lab space.

Fast cutting.

For vibrating blade tissue slicers, it takes 10 to 30 seconds to cut a slice. The VF-200 and VF-300 takes less than 5 second to cut a slice.

Simple.

The operation of the VF-200 and VF-300 are simpler than that of other vibrating slicers. Close monitoring is unnecessary. No experience is required to operate the device.

Immunohistochemistry

The Compresstome™ VF-600 is specially designed for cutting fixed tissue slices for floating section immunohistochemistry. Because of itsmany advantages, it is the machine of choice for floating section studies. The Compresstome™ VF-600 can cut slices as thin as 10 microns, equivalent in thickness to 7 microns of dehydrated paraffin-embeded slices.


Advantages over paraffin microtome

Cutting with the Compresstome microtomes requires 3 simple steps. Fix the tissue, embed in agarose, and cut.

Save Time: It saves you 80% of the time.

Saves material: You can skip 6 procedures: no dehydration/hydration, don't need to deal with waxes, or with antigen retrieval.

Better data: Less treatment means higher quality and more dependable data.

Slices in under an hour: Combined with ultra-rapid microwave fixation, the VF-600 can produce histology slices for surgical pathology within an hour.

 


Advantages over cryostat

Simpler Protocol

There are only three steps for the Compresstome™ protocol --tissue fixation, tissue slicing, and free floating staining. No over night sucrose incubation, no OCT compound embedding, no liquid nitrogen freezing. This saves you time and money.

Stronger signals

The Compresstome™eliminates the freeze-thaw step(s) that can lead to degradation in proteins and their antigenicities. This reduces false negative results for low level signals.

Better histology image

There is no ice crystal formation with the Compresstome™ slice. Cellular structure and histology image are better preserved. Further EM study is possible.

Wider cutting window

The cyrostat can slice tissues between 4 to 40 microns. For experiments requiring thicker slices, the cryostat slices cracks easily. The Compresstome™can cut slice thickness from 8 micron and up, with no upper thickness limit.

Versatility

Compresstome™ can cut fresh tissue slices for tissue culture, or for brain slice recording.

Convenient

The footprint of the Compresstome™ is very small. It can be easily moved around from counter to counter.

Affordable, &, lt;, /o:p>

The total cost for the Compresstome™ is only a small fraction of that of a cryostat. The slicer itself is much cheaper. The savings on material cost and time is even bigger.

Compresstome™ VF-600:

The compresstome™ VF-600 is our newest fixed tissue slicer. It is designed for histologists who need higher quality tissue slices in their experiments.
The VF-600 microtome integrates new structural design and stablization techniques that further innovates on the basis from our previous generation of fixed tissue microtomes.
The compresstome™ VF-600 is mainly used for free-floating immunohistochemistry and free-floating in situ hybridization experiments.

Technical specs

* Advance Speed: 0-20 mm / s, adjustable
* Return Speed: 20 mm / s, fixed
* Vibration Frequency: 0-20 Hz, adjustable
* Vibration Amplitude: 0.6 mm, fixed
* Z-axis Vibration: < 1µm
* Blade: double edge razor blade
* Cutting Angle: 13 o fixed (standard)
* Thickness Adjustment: digital adjustment, automatic
* Minimum Slice Thickness: 7µm
* Maximum Adjustable Thickness: 250 µm
* Slice Thickness Resolution: 1 µm
* Maximum Tissue Diameter: 12.5 mm (standard tube), 15.5 mm (large tube)
* Specimen Compression Ratio: 10%
* Maximum Tissue Length: 25 mm
* Cutting Mode: single/continue selectable
* Bath: 120 x 60 x 30 mm
* Power Source: DC 13-15 V
* Power Consumption: 4 W
* Dimension ( L x W x H ): 330 x 240 x 190 mm
* Weight: 7 kg

 

"...When I use the VF-200, I have significantly greater number of surface neurons..."

-Dr. Newton Woo, NICHD/NIH

"...The cell quality was very good and the field was very clean..."

-Dr. Jiang-Hong Ye, UMDNJ

 

Live tissues for electrophysiology (P12 rats, 250 µM):

Cortex (Click on picture to enlarge)

Slicer Images Slicer Images Slicer Images

Hippocampus (Click on picture to enlarge)

Slicer Images Slicer Images Slicer Images

Hypothalamus (Click on picture to enlarge)

Slicer Images

PAG (Click on picture to enlarge)

Slicer Images Slicer Images

VTA (Click on picture to enlarge)

Slicer Images Slicer Images Slicer Images Slicer Images Slicer Images Slicer Images

Live tissues for electrophysiology (P32 rats, 400 µM):

Hippocampus (Click on picture to enlarge)

Slicer Images Slicer Images

Hypothalamus (Click on picture to enlarge)

Slicer Images

PAG (click on picture to enlarge)

Slicer Images Slicer Images

Fixed tissues for immunohistochemistry:

Rat Frontal Cortex, SII receptor (Click on picture to enlarge)

Slicer Images

Rat Kidney, SII receptor (Click on picture to enlarge)

Slicer Images

Rat Liver, SII receptor (Click on picture to enlarge)

Slicer Images

Zebra Finch RA nucleus, CB1 receptor (Click on picture to enlarge)

Slicer Images

 

What People Are Saying?

Newton Woo, PhD NICHD/NIH

"For the past several months, I have been using the Precisionary Instruments Inc VF-200 to cut 300 um prefrontal cortical slices for electrophysiological recordings. As a result, I have been consistently cutting exceptional quality brain slices and have succeeded in obtaining stable whole-cell patch recordings from numerous neurons. Because I am using transgenic mice that have fluorescent reporters, it is of great importance to achieve maximal cell viability and minimal cell death. When I use the VF-200, I have significantly greater number of surface neurons than those slices cut from a regular Vibratome™. In addition the slice surface is much more even, which allows for better imaging. A small adjustment I have made to this system is melting dental wax between the blade and the holder, which holds the blade in place sufficiently, instead of the conventional method using glue. This alleviates many of the problems associated with removing the glue off the blade holder. I am thoroughly impressed with the VF-200 and would not hesitate recommending this quality product to fellow electrophysiologists. Shown below is a sample image of a 300um PFC slice taken from a 3 week old mouse."

 

-Dr. Wu Jie, Director of Epilepsy Research, Barrow Neurological Institute.

For months we attempted to obtain mechanically-dissociated neurons with adherent boutons but had no success. It was determined that perhaps our choice of vibratome was impeding the obtainment of our goals. At first, we used a microtome purchased from World Precision Instruments, and after repeated failure we purchased a tissue sectioning system from The Vibratome Company, but again we experienced continual failure. Then, we became familiar with a microtome manufactured by Precision Instruments, Inc (VF-200). Immediately we experienced success in obtaining mechanically-dissociated neurons with adherent pre-synaptic boutons using the microtome from Precisionary Instruments, Inc., and we also noticed cell quality was much higher when compared to neurons obtained using the other vibratomes. Also, cell survival rates were significantly higher when using the VF-200. Currently, we are able to routinely perform (on a daily basis) patch-clamp recordings from mechanically-dissociated neurons with adherent boutons only when using the VF-200, which was previously unobtainable using vibratomes purchased from other leading companies. As a result, we are able to successfully study pre-synaptic receptor function using single, dissociated neurons and help elucidate the mechanisms governing nicotine-induced reward and dependence. A sample of our results obtained using the VF-200 is provided below.

Fig. 1. Mechanical dissociation (with no enzyme treatment) of single neurons from rat VTA. From the VTA region (Aa), single neurons were mechanically isolated (Ab-e, phase contrast pictures) using the nerve-adherent-bouton technique. B shows an isolated VTA neuron following patch-clamp recording (Ba) labeled with lucifer yellow (Bb) and exhibiting a positive reaction to tyrosine hydroxylase (Bc), confirming its dopaminergic (DAergic) phenotype. Using the fluorescent dye FM1-43, pre-synaptic boutons adherent on a mechanically-dissociated VTA DAergic neuron were revealed (C, yellow arrows). Scale bars = 30 ?m in A-C.

Fig. 2. Pharmacological characterization of sIPSCs in mechanically-dissociated VTA DAergic neurons. The solid, horizontal bars in Aa,c,e indicate time periods of drug exposure. Ab,d,f reveal frequency of sIPSCs. B: Summary of pharmacological effects on sIPSCs.

-Dr. Jiang-Hong Ye, Anesthesiology, Pharmacology and Physiology, University of Medicine and Dentistry of New Jersey

"I was lucky to be the first one to try the VF-200 made by Precisionary Instruments Inc. At first, I was skeptical, but the results were astonishing. I have been using a Vibroslice ™ (Campden Instruments, Leicester , UK) for over 13 years, and I would never get so many living cells in one field as with the VF-200. This is also true and especially impressive for tissue of older animals. The oldest rats we used were 31 days of age. The cell quality was very good and the field was very clean. The VF-200 has greatly improved our productivity. Many thanks to Precisionary Instruments Inc. for creating such a wonderful instrument, and for their significant contribution to the neuro-science field."



VF-200 References

* Ulrike Meyer, Jing Shao, Saurish Chakrabarty, Sebastian F. Brandt, Harald Luksch and Ralf Wessel : Distributed delays stabilize neural feedback systems Biological Cybernetics Volume 99, Number 1 / July, 2008 P.79-87

* Hsieh CY, Hong CT, Cramer KS.: Deletion of EphA4 enhances deafferentation-induced ipsilateral sprouting in auditory brainstem projections. J Comp Neurol. 2007 Oct 10;504(5):508-18.

* Li KY, Xiao C, Xiong M, Delphin E, Ye JH.: Nanomolar propofol stimulates glutamate transmission to dopamine neurons: a possible mechanism of abuse potential? J Pharmacol Exp Ther. 2008 Jan 23;

* Ye JH, Ren J.: Cocaine inhibition of GABA(A) current: role of dephosphorylation. Crit Rev Neurobiol. 2006;18(1-2):85-94.

* Xiao C, Zhang J, Krnjevic' K, Ye JH.: Effects of ethanol on midbrain neurons: role of opioid receptors. Alcohol Clin Exp Res. 2007 Jul;31(7):1106-13.

* Xiao C, Zhou C, Li K, Ye JH.: Presynaptic GABAA receptors facilitate GABAergic transmission to dopaminergic neurons in the ventral tegmental area of young rats. J Physiol. 2007 May 1;580(Pt.3):731-43.

* Ye JH, Zhang J, Xiao C, Kong JQ.: Patch-clamp studies in the CNS illustrate a simple new method for obtaining viable neurons in rat brain slices: glycerol replacement of NaCl protects CNS neurons. J Neurosci Methods. 2006 Dec 15;158(2):251-9.