Japan (JA)
国・地域を選択してください。
1500個の分子と1475個の分子を区別することができるか？
本ガイドの対象者 :
顕微鏡カメラを使用している、またはこれから使用したいと考えている研究者の方
本ガイドの内容 :
カメラのスペックと実験との関連性について説明します。
セクション :
仕様の表記について :
| 浜松ホトニクスの表記 | 他メーカーの表記 |
|---|---|
| 飽和電荷量 | Pixel well depth |
| ダイナミックレンジ | Dynamic range |
| デジタル出力 | A/D conversion factor, Digitization, Digital output format |
多くの科学的な質問と同様に、「1500個の分子と1475個の分子を区別できるだろうか」という一見単純な質問に対する答えは、一筋縄には回答できせん。蛍光体の光子放出特性、分子あたりの蛍光体の数、そしてカメラに依存します。この問題の核心は、特定の実験に対してカメラがその信号を検出できるかどうかを理解することにあります。そして、検出可能な信号変化を理解するためには、飽和電荷量を見る必要があります。
検出可能な信号変化（Detectable Signal Change、以降DSC）とは、変化に対する感度を表します。顕微鏡カメラの場合、DSCは信号の変化の測定精度を表し、獲得した信号の総量が制限要因になります。カメラが検出できる最大信号量を示す飽和電荷量が最も重要であり、カメラのノイズはほとんど重要ではありません。
1500分子の検出と1475分子の検出という問題に戻ると、"2%の差を検出できるか "という問題になります。DSCと％DSCについて以下の式を使用します。
これをSについて解くと、
つまり、1500個、1475個の分子を区別するためには、飽和する前に2500個の電子を溜めることのできる十分な飽和電荷量がカメラに必要です。
もちろん、試料は、露光時間とシステムの量子効率によって割り当てられた時間内に、検出器内でそれだけの電子を生成するのに十分な光子を生成する必要があります。
しかし、この2％の差を検出しながら、画像の他の特徴も可視化（ノイズ以上）し、飽和させないようにしたいとしたらどうでしょうか。例えば、神経細胞を撮影する場合、細胞体は明るく、樹状突起はかなり暗いというような場合です。ここで、カメラのダイナミックレンジ (DR) を考慮する必要があります。
カメラのDRは、飽和電荷量の関数でもありますが、ここではカメラのノイズを考慮することが重要です。DRはサンプルを考慮せず、あくまでカメラの特性であることに注意してください。
DRはカメラが検出可能な最大信号 (S) をカメラノイズ (N) で割ったものとして定義されます。
比率ではなくデシベルで表記されることもあります。
カメラが検出可能な最大信号は、飽和電荷量によって決まります。飽和電荷量が30,000電子であれば、その画素の1画像で検出できる光子の最大数は30,000です。もちろん、すべての光子が電子に変換されるわけではないので、これは実際の光子の数のほんの一部しか反映しておらず、その特定の波長におけるカメラの量子効率 (QE) で表されます。
そしてノイズは、読み出しノイズ、暗電流ノイズ、光ショットノイズの組み合わせです (詳しくはこちら) 。一般的に、飽和電荷量と読み出しノイズを使用してダイナミックレンジを計算することができます。ほとんどの生物学的実験では、暗電流ノイズは無視でき、5分以上の露光時にのみダイナミックレンジに影響を与えます。また信号量が多い場合の光ショットノイズも無視できます。
ORCAⓇ -Flash4.0を例にとると、飽和電荷量30,000e-をスロースキャン時の読み出しノイズ0.9 electronで割ると、ダイナミックレンジは33000：1、つまり90.4Dbとなります。
尚、EM-CCDカメラの場合は、EMゲインを使用するとダイナミックレンジが狭くなることがあります。EMゲインレジスタの飽和電荷量が、個々の画素の増幅された飽和電荷量を超えると、ダイナミックレンジは減少します。その結果、高いEMゲインと広いダイナミックレンジはトレードオフの関係になります。
ビット深度について
ビット深度とダイナミックレンジは同義ではないことを認識することが重要です。飽和電荷量をビット深度で割ると、各グレーレベルの精度の限界となり、グレーレベル (デジタイザビット) あたり何個の電子が必要かという値となる。
定量的な画像解析や特徴の分類には、高いビット深度が必要不可欠です。
まとめ :
- ダイナミックレンジは、カメラがいくつの異なる強度レベルを区別できるかを示すものですが、実際に検出可能な信号変化は、サンプルの強度とカメラに依存します。
- メーカーがダイナミックレンジを記載していない場合は、飽和電荷量と読み出しノイズから計算することができます。
- EM-CCDではEMゲインを上げると、ダイナミックレンジは減少します。
- Confirmation
-
It looks like you're in the . If this is not your location, please select the correct region or country below.
You're headed to Hamamatsu Photonics website for JP (Japanese). If you want to view an other country's site, the optimized information will be provided by selecting options below.
In order to use this website comfortably, we use cookies. For cookie details please see our cookie policy.
- Cookie Policy
-
This website or its third-party tools use cookies, which are necessary to its functioning and required to achieve the purposes illustrated in this cookie policy. By closing the cookie warning banner, scrolling the page, clicking a link or continuing to browse otherwise, you agree to the use of cookies.
Hamamatsu uses cookies in order to enhance your experience on our website and ensure that our website functions.
You can visit this page at any time to learn more about cookies, get the most up to date information on how we use cookies and manage your cookie settings. We will not use cookies for any purpose other than the ones stated, but please note that we reserve the right to update our cookies.
1. What are cookies?
For modern websites to work according to visitor’s expectations, they need to collect certain basic information about visitors. To do this, a site will create small text files which are placed on visitor’s devices (computer or mobile) - these files are known as cookies when you access a website. Cookies are used in order to make websites function and work efficiently. Cookies are uniquely assigned to each visitor and can only be read by a web server in the domain that issued the cookie to the visitor. Cookies cannot be used to run programs or deliver viruses to a visitor’s device.
Cookies do various jobs which make the visitor’s experience of the internet much smoother and more interactive. For instance, cookies are used to remember the visitor’s preferences on sites they visit often, to remember language preference and to help navigate between pages more efficiently. Much, though not all, of the data collected is anonymous, though some of it is designed to detect browsing patterns and approximate geographical location to improve the visitor experience.
Certain type of cookies may require the data subject’s consent before storing them on the computer.
2. What are the different types of cookies?
This website uses two types of cookies:
- First party cookies. For our website, the first party cookies are controlled and maintained by Hamamatsu. No other parties have access to these cookies.
- Third party cookies. These cookies are implemented by organizations outside Hamamatsu. We do not have access to the data in these cookies, but we use these cookies to improve the overall website experience.
3. How do we use cookies?
This website uses cookies for following purposes:
- Certain cookies are necessary for our website to function. These are strictly necessary cookies and are required to enable website access, support navigation or provide relevant content. These cookies direct you to the correct region or country, and support security and ecommerce. Strictly necessary cookies also enforce your privacy preferences. Without these strictly necessary cookies, much of our website will not function.
- Analytics cookies are used to track website usage. This data enables us to improve our website usability, performance and website administration. In our analytics cookies, we do not store any personal identifying information.
- Functionality cookies. These are used to recognize you when you return to our website. This enables us to personalize our content for you, greet you by name and remember your preferences (for example, your choice of language or region).
- These cookies record your visit to our website, the pages you have visited and the links you have followed. We will use this information to make our website and the advertising displayed on it more relevant to your interests. We may also share this information with third parties for this purpose.
Cookies help us help you. Through the use of cookies, we learn what is important to our visitors and we develop and enhance website content and functionality to support your experience. Much of our website can be accessed if cookies are disabled, however certain website functions may not work. And, we believe your current and future visits will be enhanced if cookies are enabled.
4. Which cookies do we use?
There are two ways to manage cookie preferences.
- You can set your cookie preferences on your device or in your browser.
- You can set your cookie preferences at the website level.
If you don’t want to receive cookies, you can modify your browser so that it notifies you when cookies are sent to it or you can refuse cookies altogether. You can also delete cookies that have already been set.
If you wish to restrict or block web browser cookies which are set on your device then you can do this through your browser settings; the Help function within your browser should tell you how. Alternatively, you may wish to visit www.aboutcookies.org, which contains comprehensive information on how to do this on a wide variety of desktop browsers.
5. What are Internet tags and how do we use them with cookies?
Occasionally, we may use internet tags (also known as action tags, single-pixel GIFs, clear GIFs, invisible GIFs and 1-by-1 GIFs) at this site and may deploy these tags/cookies through a third-party advertising partner or a web analytical service partner which may be located and store the respective information (including your IP-address) in a foreign country. These tags/cookies are placed on both online advertisements that bring users to this site and on different pages of this site. We use this technology to measure the visitors' responses to our sites and the effectiveness of our advertising campaigns (including how many times a page is opened and which information is consulted) as well as to evaluate your use of this website. The third-party partner or the web analytical service partner may be able to collect data about visitors to our and other sites because of these internet tags/cookies, may compose reports regarding the website’s activity for us and may provide further services which are related to the use of the website and the internet. They may provide such information to other parties if there is a legal requirement that they do so, or if they hire the other parties to process information on their behalf.
If you would like more information about web tags and cookies associated with on-line advertising or to opt-out of third-party collection of this information, please visit the Network Advertising Initiative website http://www.networkadvertising.org.
6. Analytics and Advertisement Cookies
We use third-party cookies (such as Google Analytics) to track visitors on our website, to get reports about how visitors use the website and to inform, optimize and serve ads based on someone's past visits to our website.
You may opt-out of Google Analytics cookies by the websites provided by Google:
https://tools.google.com/dlpage/gaoptout?hl=en
As provided in this Privacy Policy (Article 5), you can learn more about opt-out cookies by the website provided by Network Advertising Initiative:
http://www.networkadvertising.org
We inform you that in such case you will not be able to wholly use all functions of our website.
Close